U.S. patent application number 16/108624 was filed with the patent office on 2019-07-11 for two-stage thermal convection apparatus and uses thereof.
This patent application is currently assigned to Ahram Biosystems, Inc.. The applicant listed for this patent is Ahram Biosystems, Inc.. Invention is credited to Hyun Jin Hwang.
Application Number | 20190210028 16/108624 |
Document ID | / |
Family ID | 44304736 |
Filed Date | 2019-07-11 |
View All Diagrams
United States Patent
Application |
20190210028 |
Kind Code |
A1 |
Hwang; Hyun Jin |
July 11, 2019 |
TWO-STAGE THERMAL CONVECTION APPARATUS AND USES THEREOF
Abstract
Disclosed is a multi-stage thermal convection apparatus such as
a two-stage thermal convection apparatus and uses thereof. In one
embodiment, the two-stage thermal convection apparatus includes a
temperature shaping element that assists a thermal convection
mediated Polymerase Chain Reaction (PCR). The invention has a wide
variety of applications including amplifying nucleic acid without
cumbersome and expensive hardware associated with many prior
devices. In a typical embodiment, the apparatus can fit in the palm
of a user's hand for use as a portable, simple to operate, and low
cost PCR amplification device.
Inventors: |
Hwang; Hyun Jin; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ahram Biosystems, Inc. |
Seoul |
|
KR |
|
|
Assignee: |
Ahram Biosystems, Inc.
Seoul
KR
|
Family ID: |
44304736 |
Appl. No.: |
16/108624 |
Filed: |
August 22, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15398618 |
Jan 4, 2017 |
10086375 |
|
|
16108624 |
|
|
|
|
13539765 |
Jul 2, 2012 |
9573133 |
|
|
15398618 |
|
|
|
|
PCT/IB2011/050104 |
Jan 11, 2011 |
|
|
|
13539765 |
|
|
|
|
61294446 |
Jan 12, 2010 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/50825 20130101;
B01L 2200/142 20130101; B01L 2300/1822 20130101; B01L 2300/1805
20130101; B01L 2400/0409 20130101; B01L 2300/042 20130101; B01L
2300/0654 20130101; B01L 2300/0861 20130101; B01L 7/52 20130101;
B01L 2200/147 20130101; B01L 2300/1838 20130101; B01L 2300/1883
20130101 |
International
Class: |
B01L 7/00 20060101
B01L007/00; B01L 3/00 20060101 B01L003/00 |
Claims
1. An apparatus adapted to perform thermal convection PCR
comprising: (a) a first heat source for heating or cooling a
channel and comprising a top surface and a bottom surface, the
channel being adapted to receive a reaction vessel for performing
PCR, (b) a second heat source for heating or cooling the channel
and comprising a top surface and a bottom surface, the bottom
surface facing the top surface of the first heat source, wherein
the channel is defined by a bottom end contacting the first heat
source and a through hole contiguous with the top surface of the
second heat source, and further wherein center points between the
bottom end and the through hole form a channel axis about which the
channel is disposed, (c) at least one temperature shaping element
such as at least one protrusion in at least one of the first and
second heat sources, the protrusion being disposed about the
channel axis and extending toward the other heat source or away
from the top or bottom surface of the heat source that comprises
the protrusion; and (d) a receptor hole adapted to receive the
channel within the first heat source.
2. The apparatus of claim 1, wherein the apparatus comprises a
first insulator positioned between the top surface of the first
heat source and the bottom surface of the second heat source.
3. The apparatus of claim 1, wherein the apparatus comprises a
first chamber disposed around the channel and within at least part
of the second or first heat source, the first chamber comprising a
first chamber top end facing a first chamber bottom end along the
channel axis and at least one chamber wall disposed around the
channel axis.
4. The apparatus of claim 3, wherein the first chamber is
positioned within the second heat source and the apparatus further
comprises a second chamber positioned in the second heat
source.
5-12. (canceled)
13. The apparatus of claim 2, wherein the first insulator comprises
a solid or a gas.
14. The apparatus of claim 3, wherein the first chamber comprises a
solid or a gas.
15. (canceled)
16. The apparatus of any of claims 13-14, wherein the gas is
air.
17-35. (canceled)
36. The apparatus of claim 3, wherein the first chamber is disposed
essentially symmetrically about the channel along a plane
perpendicular to the channel axis.
37. The apparatus of claim 3, wherein at least part of the first
chamber is disposed asymmetrically about the channel along a plane
perpendicular to the channel axis.
38-49. (canceled)
50. The apparatus of claim 4, wherein the first chamber is spaced
from the second chamber by a length (l) along the channel axis.
51. The apparatus of claim 50, wherein the first chamber, the
second chamber, and the second heat source define a first thermal
brake contacting the channel between the first and second chambers
with an area and a thickness (or a volume) sufficient to reduce
heat transfer from the first heat source.
52-53. (canceled)
54. The apparatus of claim 4, wherein the apparatus comprises a
first insulator positioned between the top surface of the first
heat source and the bottom surface of the second heat source, and
the first chamber and the first insulator define a first thermal
brake contacting the channel between the first chamber and the
first insulator with an area and a thickness (or a volume)
sufficient to reduce heat transfer from the first heat source.
55-58. (canceled)
59. The apparatus of claim 1, wherein the second heat source
comprises at least one protrusion extending away from the second
heat source toward the first heat source or away from the top
surface of the second heat source.
60-63. (canceled)
64. The apparatus of claim 1, wherein the first heat source
comprises at least one protrusion extending away from the first
heat source toward the second heat source or away from the bottom
surface of the first heat source.
65-69. (canceled)
70. The apparatus of claim 1, wherein the apparatus is adapted so
that the channel axis is tilted with respect to the direction of
gravity.
71. The apparatus of claim 70, wherein the channel axis is
perpendicular to the top or bottom surface of any of the first and
second heat sources, and the apparatus is tilted.
72. The apparatus of claim 70, wherein the channel axis is tilted
from a direction perpendicular to the top or bottom surface of any
of the first and second heat sources.
73-150. (canceled)
151. The apparatus of claim 1, wherein the apparatus is adapted to
generate a centrifugal force inside the channel so as to modulate
the convection PCR.
152-162. (canceled)
163. A PCR centrifuge adapted to perform a polymerase chain
reaction (PCR) under centrifugation conditions, the PCR centrifuge
comprising the apparatus featured in claim 151.
164. A method for performing a polymerase chain reaction (PCR) by
thermal convection, the method comprising at least one and
preferably all of the following steps: (a) maintaining a first heat
source comprising a receptor hole at a temperature range suitable
for denaturing a double-stranded nucleic acid molecule and forming
a single-stranded template, (b) maintaining a second heat source at
a temperature range suitable for annealing at least one
oligonucleotide primer to the single-stranded template; and (c)
producing thermal convection between the receptor hole and the
second heat source under conditions sufficient to produce the
primer extension product, wherein a channel that is adapted to
receive a reaction vessel for performing the PCR is defined by a
bottom end of the receptor hole contacting the first heat source
and a through hole contiguous with the top surface of the second
heat source, and further wherein center points between the bottom
end of the receptor hole and the through hole form a channel axis
about which the channel is disposed; and wherein the method further
comprising a step of providing at least one protrusion in at least
one of the first and second heat sources, the protrusion being
disposed about the channel axis and extending toward the other heat
source or away from the top or bottom surface of the heat source
that comprises the protrusion.
165. The method of claim 164, wherein the method further comprises
a step of providing the reaction vessel comprising the
double-stranded nucleic acid molecule and the oligonucleotide
primer in aqueous solution, and a DNA polymerase in aqueous
solution or an immobilized DNA polymerase.
166-167. (canceled)
168. The method of claim 165, wherein the method further comprises
a step of contacting the reaction vessel to the receptor hole and a
chamber disposed within at least one of the second or first heat
source, the contacting being sufficient to support the thermal
convection within the reaction vessel.
169. The method of claim 168, wherein the method further comprises
a step of contacting the reaction vessel to a first insulator
between the first and second heat sources.
170-171. (canceled)
172. The method of claim 165, wherein the method further comprises
a step of producing a fluid flow within the reaction vessel that is
essentially symmetric about the channel axis.
173. The method of claim 165, wherein the method further comprises
a step of producing a fluid flow within the reaction vessel that is
asymmetric about the channel axis.
174. The method of claim 165, wherein at least steps (a)-(b)
consume less than about 1 W of power per reaction vessel to produce
the primer extension product.
175-178. (canceled)
179. The method of claim 164, wherein the method further comprises
a step of applying a centrifugal force to the reaction vessel
conducive to performing the PCR.
180. A method for performing a polymerase chain reaction (PCR) by
thermal convection, the method comprising the steps of adding an
oligonucleotide primer, nucleic acid template, DNA polymerase, and
buffer to a reaction vessel received by the apparatus of claim 1
under conditions sufficient to produce a primer extension
product.
181. (canceled)
182. A method for performing a polymerase chain reaction (PCR) by
thermal convection, the method comprising the steps of adding an
oligonucleotide primer, nucleic acid template, DNA polymerase, and
buffer to a reaction vessel received by the PCR centrifuge of claim
163 and applying a centrifugal force to the reaction vessel under
conditions sufficient to produce a primer extension product.
209. The apparatus of any of claims 1 and 151 further comprising at
least one optical detection unit.
210. The PCR centrifuge of claim 163, further comprising at least
one optical detection unit.
211. The method of any one of claims 164 and 179, further
comprising the step of detecting the primer extension product in
real-time by using at least one optical detection unit.
212. The method of any of claims 180 and 182, further comprising
the step of detecting the primer extension product in real-time by
using at least one optical detection unit.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation-in-part
application of PCT/IB2011/050104, filed on Jan. 11, 2011 which
claims priority to U.S. Provisional Application No. 61/294,446 as
filed on Jan. 12, 2010, the disclosure of which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention features a multi-stage thermal
convection apparatus, particularly a two-stage thermal convection
apparatus and uses thereof. The apparatus includes at least one
temperature shaping element that assists a polymerase chain
reaction (PCR). The invention has a wide variety of applications
including amplifying a DNA template without the cumbersome and
often expensive hardware associated with prior devices. In one
embodiment, the apparatus can fit in the palm of a user's hand for
use as a portable PCR amplification device.
BACKGROUND
[0003] The polymerase chain reaction (PCR) is a technique that
amplifies a polynucleotide sequence each time a temperature
changing cycle is completed. See for example, PCR: A Practical
Approach, by M. J. McPherson, et al., IRL Press (1991), PCR
Protocols: A Guide to Methods and Applications, by Innis, et al.,
Academic Press (1990), and PCR Technology: Principals and
Applications for DNA Amplification, H. A. Erlich, Stockton Press
(1989). PCR is also described in many patents, including U.S. Pat.
Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818;
5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and
5,066,584.
[0004] In many applications, PCR involves denaturing a
polynucleotide of interest ("template"), followed by annealing a
desired primer oligonucleotide ("primer") to the denatured
template. After annealing, a polymerase catalyzes synthesis of a
new polynucleotide strand that incorporates and extends the primer.
This series of steps: denaturation, primer annealing, and primer
extension, constitutes a single PCR cycle. These steps are repeated
many times during PCR amplification.
[0005] As cycles are repeated, the amount of newly synthesized
polynucleotide increases geometrically. In many embodiments,
primers are selected in pairs that can anneal to opposite strands
of a given double-stranded polynucleotide. In this case, the region
between the two annealing sites can be amplified.
[0006] There is a need to vary the temperature of the reaction
mixture during a multi-cycle PCR experiment. For example,
denaturation of DNA typically takes place at about 90.degree. C. to
about 98.degree. C. or a higher temperature, annealing a primer to
the denatured DNA is typically performed at about 45.degree. C. to
about 65.degree. C., and the step of extending the annealed primers
with a polymerase is typically performed at about 65.degree. C. to
about 75.degree. C. These temperature steps must be repeated,
sequentially, for PCR to progress optimally.
[0007] To satisfy this need, a variety of commercially available
devices has been developed for performing PCR. A significant
component of many devices is a thermal "cycler" in which one or
more temperature controlled elements (sometimes called "heat
blocks") hold the PCR sample. The temperature of the heat block is
varied over a time period to support the thermal cycling.
Unfortunately, these devices suffer from significant
shortcomings.
[0008] For example, most of the devices are large, cumbersome, and
typically expensive. Large amounts of electric power are usually
required to heat and cool the heat block to support the thermal
cycling. Users often need extensive training. Accordingly, these
devices are generally not suitable for field use.
[0009] Attempts to overcome these problems have not been entirely
successful. For instance, one attempt involved use of multiple
temperature controlled heat blocks in which each block is kept at a
desired temperature and sample is moved between heat blocks.
However, these devices suffer from other drawbacks such as the need
for complicated machinery to move the sample between different heat
blocks and the need to heat or cool one or a few heat blocks at a
time.
[0010] There have been some efforts to use thermal convection in
some PCR processes. See Krishnan, M. et al. (2002) Science 298:
793; Wheeler, E. K. (2004) Anal. Chem. 76: 4011-4016; Braun, D.
(2004) Modern Physics Letters 18: 775-784; and WO02/072267.
However, none of these attempts has produced a thermal convection
PCR device that is compact, portable, more affordable and with a
less significant need for electric power. Moreover, such thermal
convection devices often suffer from low PCR amplification
efficiency and limitation in the size of amplicon.
SUMMARY
[0011] The present invention provides a multi-stage thermal
convection apparatus, particularly a two-stage thermal convection
apparatus and uses thereof. The apparatus generally includes at
least one temperature shaping element to assist a polymerase chain
reaction (PCR). As described below, a typical temperature-shaping
element is a structural and/or positional feature of the apparatus
that supports thermal convection PCR. Presence of the temperature
shaping element enhances the efficiency and speed of the PCR
amplification, supports miniaturization, and reduces need for
significant power. In one embodiment, the apparatus readily fits in
the palm of a user's hand and has low power requirements sufficient
for battery operation. In this embodiment, the apparatus is
smaller, less expensive and more portable than many prior PCR
devices.
[0012] Accordingly, and in one aspect, the present invention
features a two-stage thermal convection apparatus adapted to
perform thermal convection PCR amplification ("apparatus").
Preferably, the apparatus has at least one of and preferably all of
the following elements as operably linked components: [0013] (a) a
first heat source for heating or cooling a channel and comprising a
top surface and a bottom surface, the channel being adapted to
receive a reaction vessel for performing PCR, [0014] (b) a second
heat source for heating or cooling the channel and comprising a top
surface and a bottom surface, the bottom surface facing the top
surface of the first heat source, wherein the channel is defined by
a bottom end contacting the first heat source and a through hole
contiguous with the top surface of the second heat source, and
further wherein center points between the bottom end and the
through hole form a channel axis about which the channel is
disposed, [0015] (c) at least one temperature shaping element
adapted to assist thermal convection PCR; and [0016] (d) a receptor
hole adapted to receive the channel within the first heat
source.
[0017] Also provided is a method of making the forgoing apparatus
which method includes assembling each of (a)-(d) in an operable
combination sufficient to perform thermal convection PCR as
described herein.
[0018] In another aspect of the present invention, there is
provided a thermal convection PCR centrifuge ("PCR centrifuge")
adapted to perform PCR using at least one of the apparatus as
described herein.
[0019] Further provided by the present invention is a method for
performing a polymerase chain reaction (PCR) by thermal convection.
In one embodiment, the method includes at least one of and
preferably all of the following steps: [0020] (a) maintaining a
first heat source comprising a receptor hole at a temperature range
suitable for denaturing a double-stranded nucleic acid molecule and
forming a single-stranded template, [0021] (b) maintaining a second
heat source at a temperature range suitable for annealing at least
one oligonucleotide primer to the single-stranded template, and
[0022] (c) producing thermal convection between the receptor hole
and the second heat source under conditions sufficient to produce
the primer extension product.
[0023] In another aspect, the invention provides reaction vessels
adapted to be received by an apparatus of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a schematic drawing showing an overhead view of an
embodiment of the apparatus. Sectional planes through the apparatus
(A-A and B-B) are depicted.
[0025] FIGS. 2A-C are schematic drawings showing sectional views of
an apparatus embodiment having a first chamber 100. FIGS. 2A-C are
cross-sectional views taken along the A-A (FIGS. 2A, 2B) and B-B
planes (FIG. 2C).
[0026] FIGS. 3A-B are schematic drawings showing sectional views of
apparatus embodiments taken along the A-A plane. Each apparatus has
a first 100 and a second 110 chamber of unequal widths with respect
to the channel axis 80.
[0027] FIGS. 4A-B are schematic drawings showing a sectional view
(A-A) of an embodiment of the apparatus. FIG. 4B shows an expanded
view of the region (identified by the dotted circle in FIG. 4A).
The apparatus has a first 100 and a second 110 chamber. A region
between the first and second chambers includes a first thermal
brake 130.
[0028] FIGS. 5A-C are schematic drawings showing sectional views of
an apparatus embodiment. FIGS. 5A-C are cross-sectional views taken
along the A-A (FIGS. 5A-B) and B-B planes (FIG. 5C). The second
heat source 30 comprises a first chamber 100 and a first protrusion
33 disposed symmetrically about the channel axis 80 that extend the
length of the first chamber 100. The first heat source 20 comprises
a first protrusion 23.
[0029] FIGS. 6A-C are schematic drawings of an apparatus embodiment
taken along the A-A (FIGS. 6A-B) and B-B planes (FIG. 6C). The
first 20 and second 30 heat sources include protrusions (23, 24,
33, 34) that are each positioned symmetrically about the channel
axis 80. The second heat source 30 comprises a first chamber
100.
[0030] FIGS. 7A-D are schematic drawings showing channel
embodiments of the apparatus (A-A plane).
[0031] FIGS. 8A-J are schematic drawings showing channel
embodiments of the apparatus. The plane of section is perpendicular
to the channel axis 80.
[0032] FIGS. 9A-I are drawings showing various chamber embodiments
of the apparatus. The plane of section is perpendicular to the
channel axis 80. Hatched parts represent the second or first heat
source.
[0033] FIGS. 10A-P are drawings showing various chamber and channel
embodiments of the apparatus. The plane of section is perpendicular
to the channel axis 80. Hatched parts represent the second or first
heat source.
[0034] FIGS. 11A-B are schematic drawings showing various
positioning embodiments. FIG. 11A shows a positioning embodiment of
the apparatus shown in FIG. 5A. The apparatus is tilted (by an
angle defined by .theta..sub.g) with respect to the direction of
gravity. FIG. 11B shows an apparatus embodiment in which the
channel 70 and the first chamber 100 are tilted with respect to the
direction of gravity within the second heat source 30. The
direction of gravity remains perpendicular with respect to the heat
sources.
[0035] FIGS. 12A-B are schematic drawings showing sectional views
(A-A plane) of apparatus embodiments. The first chamber 100 is
tapered.
[0036] FIGS. 13A-B are schematic drawings showing sectional views
(A-A plane) of an apparatus embodiment having a first thermal brake
130 located in between the first 100 and second 110 chambers within
the second heat source 30. The widths of the first and second
chambers are shown to be different. FIG. 13B shows an expanded view
of the region identified by the dotted circle shown in FIG. 13A to
illustrate structural details of the first thermal brake 130.
[0037] FIGS. 14A-D are schematic drawings showing sectional views
(A-A plane) of apparatus embodiments having a first thermal brake
130 located on the bottom of the first chamber 100 (i.e., on the
bottom of the second heat source 30). FIGS. 14B and D show expanded
views of the region identified by the dotted circle shown in FIGS.
14A and D, respectively, to illustrate structural details of the
first thermal brake 130. The first chamber 100 has a straight wall
in FIGS. 14A-B and a tapered wall in FIGS. 14C-D.
[0038] FIG. 15 is a schematic drawing showing a sectional view
(A-A) of one embodiment of the apparatus. The receptor hole 73 is
asymmetrically disposed around the channel axis 80 and forms a
receptor hole gap 74.
[0039] FIGS. 16A-B are schematic drawings showing sectional views
of apparatus embodiments taken along the A-A plane. The first heat
source 20 includes a receptor hole gap 74. In the embodiment shown
by FIG. 16B, the receptor hole gap 74 includes a top surface that
is inclined with respect to the channel axis 80.
[0040] FIGS. 17A-B are schematic drawings showing sectional views
of apparatus embodiments taken along the A-A plane. The first heat
source 20 features a protrusion 23 disposed asymmetrically around
the receptor hole 73. In FIG. 17A, the protrusion 23 next to the
receptor hole 73 has multiple top surfaces one of which has a
greater height and is closer to the first chamber 100. In FIG. 17B,
the protrusion 23 has one top surface that is inclined with respect
to the channel axis 80 so that one side has a greater height and is
closer to the first chamber 100 than another side opposite to the
receptor hole 73.
[0041] FIGS. 18A-D are schematic drawings showing sectional views
of apparatus embodiments taken along the A-A plane. In these
embodiments, the first 20 and second 30 heat sources feature
protrusions 23 and 33 disposed asymmetrically about the channel
axis 80. The protrusions 23 and 33 have a greater height on one
side than another side opposite to the channel axis 80. The top end
of the protrusion 23 and the bottom end of the protrusion 33 have
multiple surfaces (FIGS. 18A and 18C) or are inclined with respect
to the channel axis 80 (FIGS. 18B and 18D). In FIGS. 18A and 18B,
the first chamber 100 features a bottom end 102 in which a portion
is closer to one side of the protrusion 23 than another portion
opposite to the channel axis 80. In FIGS. 18C and 18D, the bottom
end 102 of the first chamber 100 is located essentially at a
constant distance from the top surface of the protrusion 23.
[0042] FIGS. 19A-B are schematic drawings showing sectional views
of apparatus embodiments taken along the A-A plane. In these
embodiments, the first heat source 20 features a protrusion 23
disposed symmetrically around the receptor hole 73 and the second
heat source 30 features a protrusion 33 disposed asymmetrically
about the channel axis 80. In FIG. 19A, the bottom end 102 of the
first chamber 100 features multiple surfaces so that a portion of
the bottom end 102 that is closer to one side of the protrusion 23
than another portion opposite to the channel axis 80. In FIG. 19B,
the bottom end 102 of the first chamber 100 is inclined with
respect to the channel axis 80 so that a portion of the bottom end
102 is closer to the protrusion 23 than another portion opposite to
the channel axis 80.
[0043] FIGS. 20A-C are schematic drawings showing various apparatus
embodiments. FIG. 20A shows a sectional view of an apparatus
embodiment in which the first chamber 100 is within the second heat
source 30 and is disposed asymmetrically (off-centered) about the
channel 70. FIGS. 20B-C show sectional views of an apparatus
embodiment along the A-A plane. The first chamber 100 is disposed
asymmetrically about the channel 70. As shown in FIG. 20C, the
thermal brake 130 is shown disposed asymmetrically about the
channel 70 with the wall 133 contacting the channel 70 on one
side.
[0044] FIG. 21 is a schematic drawing showing a sectional view of
an apparatus embodiment taken along the A-A plane showing the first
100 and second 110 chambers disposed asymmetrically about the
channel axis 80 within the second heat source 30.
[0045] FIG. 22 is a schematic drawing showing a sectional view
taken along the A-A plane of an apparatus embodiment in which the
first chamber 100 includes a wall 103 disposed at an angle with
respect to the channel axis 80.
[0046] FIGS. 23A-B are schematic drawings showing a sectional view
of an apparatus embodiment taken alone the A-A plane with the first
chamber 100 and the second chamber 110 within the second heat
source 30. As shown in FIG. 23B, the apparatus features a first
thermal brake 130 asymmetrically disposed about the channel 70 and
between the first 100 and second 110 chambers with the wall 133
contacting the channel 70 on one side.
[0047] FIGS. 24A-B are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30.
The first 100 and second 110 chambers are disposed asymmetrically
about the channel axis 80. In an expanded view shown in FIG. 24B,
the thermal brake 130 is shown disposed symmetrically about the
channel 70 between the first 100 and second 110 chambers. The wall
133 of the thermal brake 130 contacts the channel 70.
[0048] FIGS. 24C-D are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30.
The first 100 and second 110 chambers are disposed asymmetrically
about the channel axis 80. The width of the first chamber 100
perpendicular to the channel axis 80 is smaller than the width of
the second chamber 110 along the channel axis 80. In an expanded
view shown in FIG. 24D, the first thermal brake 130 is shown
disposed asymmetrically about the channel 70 with the wall 133
contacting the channel 70 on one side.
[0049] FIGS. 25A-B are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30.
The first 100 and second 110 chambers are disposed asymmetrically
about the channel axis 80 in opposite directions along the A-A
plane. The thermal brake 130 is shown disposed symmetrically about
the channel 70 with the wall 133 contacting the channel 70.
[0050] FIGS. 26A-B are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30.
The first 100 and second 110 chambers are disposed asymmetrically
about the channel axis 80. As shown in FIG. 26B, the first thermal
brake 130 is also disposed asymmetrically about the channel 70 with
the wall 133 contacting the channel 70 on one side.
[0051] FIGS. 26C-D are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30
and are disposed asymmetrically about the channel axis 80. As shown
in FIG. 26D, the first thermal brake 130 is also asymmetrically
disposed about the channel 70 with the wall 133 contacting the
channel 70 on one side.
[0052] FIGS. 27A-B are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30
and are disposed asymmetrically about the channel axis 80 in
opposite directions along the A-A plane. In an expanded view shown
in FIG. 27B, the first thermal brake 130 is shown disposed
asymmetrically with the wall 133 contacting the channel 70 on one
side within the first chamber 100. The second thermal brake 140 is
also shown disposed asymmetrically with the wall 143 contacting the
channel 70 on one side within the second chamber 110. The top end
131 of the first thermal brake 130 is positioned essentially at the
same height as the bottom end 142 of the second thermal brake
140.
[0053] FIGS. 27C-D are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30
and are disposed asymmetrically about the channel axis 80 in
opposite directions along the A-A plane. In an expanded view shown
in FIG. 27D, the first 130 and second 140 thermal brakes are shown
disposed asymmetrically with the walls (133, 143) each contacting
the channel 70 on one side. The top end 131 of the first thermal
brake 130 is positioned higher than the bottom end 142 of the
second thermal brake 140.
[0054] FIGS. 27E-F are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30
and are disposed asymmetrically about the channel axis 80 in
opposite directions along the A-A plane. In an expanded view shown
in FIG. 27F, the first 130 and second 140 thermal brakes are shown
disposed asymmetrically with the walls (133, 143) each contacting
the channel 70 on one side. The top end 131 of a first thermal
brake 130 is shown positioned lower than the bottom end 142 of the
second thermal brake 140.
[0055] FIGS. 28A-B are schematic drawings showing a sectional view
of an apparatus embodiment along the A-A plane in which the first
100 and second 110 chambers are within the second heat source 30
and are disposed asymmetrically about the channel axis 80. The top
end 101 of the first chamber 100 and the bottom end 112 of the
second chamber 110 are inclined (tilted) with respect to the
channel axis 80. The wall 103 of the first chamber 100, the wall
113 of the second chamber 110 are each essentially parallel to the
channel axis 80. In an expanded view shown in FIG. 28B, the first
thermal brake 130 is shown inclined (tilted) with respect to the
channel axis 80 and the wall 133 contacts the channel 70.
[0056] FIGS. 29A-D are schematic drawings showing sectional views
of apparatus embodiments along the A-A plane in which the first 100
and second 110 chambers are within the second heat source 30 and
are disposed asymmetrically about the channel axis 80. In FIGS.
29A-D, the wall 103 of the first chamber 100 and the wall 113 of
the second chamber 110 are shown inclined (tilted) with respect to
the channel axis 80. In an expanded view shown in FIG. 29B, the
thermal brake 130 is shown symmetrically disposed about the channel
70 with the wall 133 contacting the channel 70. In an expanded view
shown in FIG. 29D, the first thermal brake 130 is shown inclined
(tilted) with respect to the channel axis 80 with the wall 133
contacting the channel 70.
[0057] FIG. 30 is a schematic drawing showing an overhead view of
an embodiment of the apparatus 10 showing first securing element
200, second securing element 210, heating/cooling elements
(160a-b), and temperature sensors (170a-b). Various sectional
planes are indicated (A-A, B-B, and C-C).
[0058] FIGS. 31A-B are schematic drawings of cross-sectional views
of the apparatus embodiment shown in FIG. 30 taken along the A-A
(FIG. 31A) and B-B (FIG. 31B) planes.
[0059] FIG. 32 is a schematic drawing of a cross-sectional view of
the first securing element 200 taken along the C-C plane.
[0060] FIG. 33 is a schematic drawing of an overhead view of an
apparatus embodiment showing various securing elements, heat source
structures, heating/cooling elements, and temperature sensors.
[0061] FIGS. 34A-B are schematic drawings of an overhead view (FIG.
34A) and a cross-sectional view (FIG. 34B) of an apparatus
embodiment showing a first housing element 300 defining a second
310 and third 320 insulator.
[0062] FIGS. 35A-B are schematic drawings of an overhead view (FIG.
35A) and a cross-sectional view (FIG. 35B) of an apparatus
embodiment comprising a second housing element 400 and a fourth 410
and fifth 420 insulator.
[0063] FIGS. 36A-B are schematic drawings of an embodiment of a PCR
centrifuge. FIG. 36A shows an overhead view and FIG. 36B shows a
cross-sectional view taken along the A-A plane.
[0064] FIG. 37 is a schematic drawing showing a cross-sectional
view of an apparatus embodiment of the PCR centrifuge taken along
the A-A plane.
[0065] FIGS. 38A-B are schematic drawings showing an embodiment of
a PCR centrifuge comprising a first chamber. In FIG. 38A, the plane
of section along A-A is through the channel 70. In FIG. 38B, the
plane of section along B-B is through the first 200 and second 210
securing means.
[0066] FIGS. 39A-B are schematic drawings showing embodiments of a
first (FIG. 39A) and second (FIG. 39B) heat source for use in the
PCR centrifuge shown in FIGS. 38A-B. Sectional planes through the
apparatus (A-A and B-B) are indicated.
[0067] FIGS. 40A-D are schematic drawings showing a cross-sectional
view of various reaction vessel embodiments.
[0068] FIGS. 41A-J are schematic drawings showing cross-sectional
views of various reaction vessel embodiments taken perpendicular to
the reaction vessel axis 95.
[0069] FIGS. 42A-C are results of thermal convection PCR using the
apparatus of FIG. 5A showing amplification of a 349 bp sequence
from a 1 ng plasmid sample with three different DNA polymerases
from Takara Bio, Finnzymes, and Kapa Biosystems, respectively.
[0070] FIG. 43 shows results of thermal convection PCR using the
apparatus of FIG. 5A showing amplification of a 936 bp sequence
from a 1 ng plasmid sample.
[0071] FIGS. 44A-D are results of thermal convection PCR using the
apparatus of FIG. 5A showing acceleration of PCR amplification at
elevated denaturation temperatures (98.degree. C., 100.degree. C.,
102.degree. C., and 104.degree. C., respectively).
[0072] FIGS. 45A-B are results of thermal convection PCR using the
apparatus of FIG. 5A showing amplification of 479 bp GAPDH (FIG.
45A) and 363 bp .beta.-globin (FIG. 45B) sequences from 10 ng human
genome samples.
[0073] FIG. 46 shows results of thermal convection PCR using the
apparatus of FIG. 5A showing amplification of a 241 bp .beta.-actin
sequence from very low copy human genome samples.
[0074] FIG. 47 shows temperature variations of the first and second
heat sources of the apparatus of FIG. 5A as a function of time when
target temperatures were set to 98.degree. C. and 64.degree. C.,
respectively.
[0075] FIG. 48 shows power consumption of the apparatus of FIG. 5A
having 12 channels as a function of time.
[0076] FIGS. 49A-E are results of thermal convection PCR using the
apparatus of FIG. 11A showing acceleration of PCR amplification for
a 349 bp plasmid target as a function of the gravity tilting angle.
The gravity tilting angle was 0.degree., 10.degree., 20.degree.,
30.degree., and 45.degree. for FIGS. 49A-E, respectively.
[0077] FIGS. 50A-E are results of thermal convection PCR using the
apparatus of FIG. 11A showing acceleration of PCR amplification for
a 936 bp plasmid target as a function of the gravity tilting angle.
The gravity tilting angle was 0.degree., 10.degree., 20.degree.,
30.degree., and 45.degree. for FIGS. 50A-E, respectively.
[0078] FIG. 51 shows results of thermal convection PCR using the
apparatus of FIG. 11A showing amplification of various target
sequences (with size between about 150 bp to about 2 kbp) from 1 ng
plasmid samples. The gravity tilting angle was 10.degree..
[0079] FIGS. 52A-E are results of thermal convection PCR using the
apparatus of FIG. 11A showing acceleration of PCR amplification for
a 521 bp human genome target as a function of the gravity tilting
angle. The gravity tilting angle was 0.degree., 10.degree.,
20.degree., 30.degree., and 45.degree. for FIGS. 52A-E,
respectively.
[0080] FIGS. 53A-B are results of thermal convection PCR using the
apparatus of FIG. 11A showing amplification of 200 bp .beta.-globin
(FIG. 53A) and 514 bp .beta.-actin (FIG. 53B) sequences from 10 ng
human genome samples. The gravity tilting angle was 10.degree..
[0081] FIG. 54 shows results of thermal convection PCR using the
apparatus of FIG. 11A showing amplification of various target
sequences (with size between about 100 bp to about 500 bp) from 10
ng human genome and cDNA samples. The gravity tilting angle was
10.degree..
[0082] FIG. 55 shows results of thermal convection PCR using the
apparatus of FIG. 11A showing amplification of a 241 bp
.beta.-actin sequence from very low copy human genome samples when
the gravity tilting angle of 10.degree. was introduced.
[0083] FIGS. 56A-B are results of thermal convection PCR using the
apparatuses of FIGS. 5A and 20A, respectively, for amplification of
a 349 bp plasmid target. The apparatus of FIG. 5A has a symmetric
heating structure and that of FIG. 20A has an asymmetric heating
structure comprising an off-centered first chamber.
[0084] FIGS. 57A-B are results of thermal convection PCR using the
apparatuses of FIGS. 5A and 20A, respectively, for amplification of
a 241 bp human genome target. The apparatus of FIG. 5A has a
symmetric heating structure and that of FIG. 20A has an asymmetric
heating structure comprising an off-centered first chamber.
[0085] FIGS. 58A-B are results of thermal convection PCR using the
apparatuses of FIGS. 5A and 20A, respectively, for amplification of
a 216 bp human genome target. The apparatus of FIG. 5A has a
symmetric heating structure and that of FIG. 20A has an asymmetric
heating structure comprising an off-centered first chamber.
[0086] FIG. 59A-B are schematic drawings showing sectional views of
apparatus embodiments having one or more optical detection units
600-603 spaced from the first heat source 20 along the channel axis
80 and sufficient to detect a fluorescence signal from the samples
in the reaction vessels 90. The apparatus includes a single optical
detection unit 600 to detect the fluorescence signal from multiple
reaction vessels (FIG. 59A) or multiple optical detection units
601-603 (FIG. 59B) to detect the fluorescence signal from each
reaction vessel. In the embodiments shown in FIGS. 59A-B, the
optical detection unit detects the fluorescence signal from the
bottom end 92 of the reaction vessel 90. The first heat source 20
comprises an optical port 610 positioned about the channel axis 80
between the bottom end 72 of the channel 70 and the first heat
source protrusion 24 that provides a path for the excitation and
emission of light parallel to the channel axis 80 (shown as upward
and downward arrows, respectively).
[0087] FIGS. 60A-B are schematic drawings showing sectional views
of apparatus embodiments having one optical detection unit 600
(FIG. 60A) or more than one optical detection units 601-603 (FIG.
60B). Each of optical detection units 600-603 is spaced from the
second heat source 30 along the channel axis 80 sufficient to
detect a fluorescence signal from the samples located in the
reaction vessels 90. In these embodiments, a center part of a
reaction vessel cap (not shown) that typically fits to the top
opening of the reaction vessel 90 functions as an optical port for
the excitation and emission light parallel to the channel axis 80
(shown in FIGS. 60A-B as downward and upward arrows,
respectively).
[0088] FIG. 61 is a schematic drawing showing a sectional view of
an apparatus embodiment having an optical detection unit 600 spaced
from the second heat source 30. In this embodiment, the optical
port 610 is positioned in the second heat source 30 (shown as gray
rectangular boxes) and the first insulator 50 (shown as dashed
lines) along a path perpendicular to the channel axis 80 toward the
optical detection unit 600 sufficient to detect a fluorescence
signal from the side of the samples in the reaction vessels 90. The
optical port 610 provides a path for the excitation and emission
light between the reaction vessel 90 and the optical detection unit
600 (shown as left and right pointing arrows or vice versa). A side
part of the reaction vessel 90 and a portion of the first chamber
100 along the light path also function as optical port in this
embodiment.
[0089] FIG. 62 is a schematic drawing showing a sectional view of
an optical detection unit 600 positioned to detect a fluorescence
signal from the bottom end 92 of the reaction vessel 90. In this
embodiment, a light source 620, an excitation lens 630, and an
excitation filter 640 that are configured to generate an excitation
light are located along a direction at a right angle with respect
to the channel axis 80, and a detector 650, an aperture or slit
655, an emission lens 660, and an emission filter 670 that are
operable to detect an emission light are located along the channel
axis 80. A dichrocic beam-splitter 680 that transmits the
fluorescence emission and reflects the excitation light is also
shown.
[0090] FIG. 63 is a schematic drawing showing a sectional view of
an optical detection unit 600 positioned to detect a fluorescence
signal from the bottom end 92 of the reaction vessel 90. In this
embodiment, a light source 620, an excitation lens 630, and an
excitation filter 640 are positioned to generate an excitation
light along the channel axis 80. A detector 650, an aperture or
slit 655, an emission lens 660, and an emission filter 670 are
positioned to detect an emission light as located along a direction
at a right angle with respect to the channel axis 80. A dichrocic
beam-splitter 680 that transmits the excitation light and reflects
the fluorescence emission is shown.
[0091] FIGS. 64A-B are schematic drawings showing sectional views
of an optical detection unit 600 positioned to detect a
fluorescence signal from the bottom end 92 of the reaction vessel
90. In these embodiments, a single lens 635 is used to shape the
excitation light and also to detect the fluorescence emission. In
the embodiment shown in FIG. 64A, the light source 620 and the
excitation filter 640 are located along a direction at a right
angle to the channel axis 80. In the embodiment shown in FIG. 64B,
the optical elements for detecting the fluorescence emission (650,
655, and 670) are located along a direction at a right angle to the
channel axis 80.
[0092] FIG. 65 is a schematic drawing showing a sectional view of
an optical detection unit 600 positioned to detect a fluorescence
signal from the top end 91 of the reaction vessel 90. As in FIG.
62, the light source 620, the excitation lens 630, and the
excitation filter 640 are located along a direction at a right
angle to the channel axis 80, and the detector 650, the aperture or
slit 655, the emission lens 660, and the emission filter 670 are
located along the channel axis 80. Also shown in this embodiment is
a reaction vessel cap 690 sealably attached to the top end 91 of
the reaction vessel 90 and including an optical port 695 disposed
around a center point of the top end 91 of the reaction vessel 90
and for transmission of the excitation and emission light. The
optical port 695 is further defined by the upper part of the
reaction vessel cap 690 and the upper part of the reaction vessel
90 in this embodiment.
[0093] FIGS. 66A-B are schematic drawings showing sectional views
of reaction vessels 90 with reaction vessel caps 690 and optical
ports 695. The reaction vessel cap 690 is sealably attached to the
upper part of the reaction vessel 90 and the optical port 695. In
these embodiments, the bottom end 696 of the optical port 695 is
made to contact the sample when the reaction vessel 90 is sealed
with the reaction vessel cap 690. An open space 698 is provided on
the side of the bottom end 696 of the optical port 695 and the
reaction vessel cap 690 so that the sample can fill up the open
space when the reaction vessel 90 is sealed with the reaction
vessel cap 690. The sample meniscus is located higher than the
bottom end 696 of the optical port 695. In FIGS. 66A-B, the optical
port 695 is disposed around a center point of the lower part of the
reaction vessel cap 690 and is further defined by the lower part of
the reaction vessel cap 690 and the upper part of the reaction
vessel 90.
[0094] FIG. 67 is a schematic drawing showing a sectional view of a
reaction vessel 90 with an optical detection unit 600 disposed
above the reaction vessel 90. The reaction vessel 90 is sealed with
the reaction vessel cap 690 having an optical port 695 disposed
around a center point of the upper part of the reaction vessel 90
sufficient to make contact with sample. In this embodiment, the
excitation light and the fluorescence emission pass through the
optical port 695 and reach the sample or vice versa without passing
air contained inside the reaction vessel 90.
DETAILED DESCRIPTION
[0095] The following figure key may help the reader better
appreciate the invention including the Drawings and claims: [0096]
10: Apparatus embodiment [0097] 20: First heat source (bottom
stage) [0098] 21: Top surface of the first heat source [0099] 22:
Bottom surface of the first heat source [0100] 23: First heat
source protrusion (pointing toward the second heat source) [0101]
24: First heat source protrusion (pointing toward table) [0102] 30:
Second heat source (intermediate stage) [0103] 31: Top surface of
the second heat source [0104] 32: Bottom surface of the second heat
source [0105] 33: Second heat source protrusion (pointing toward
the first heat source) [0106] 34: Second heat source protrusion
(pointing away from the top of the second heat source) [0107] 50:
First insulator (or first insulating gap) [0108] 51: First
insulator chamber [0109] 70: Channel [0110] 71: Top end of the
channel/through hole [0111] 72: Bottom end of the channel [0112]
73: receptor hole [0113] 74: receptor hole gap [0114] 80: (Center)
axis of the channel [0115] 90: Reaction vessel [0116] 91: Top end
of the reaction vessel [0117] 92: Bottom end of the reaction vessel
[0118] 93: Outer wall of the reaction vessel [0119] 94: Inner wall
of the reaction vessel [0120] 95: (Center) axis of the reaction
vessel [0121] 100: First Chamber [0122] 101: Top end of the first
chamber, defining an upper limit of the chamber [0123] 102: Bottom
end of the first chamber, defining a lower limit of the chamber
[0124] 103: First wall of the first chamber, defining a horizontal
limit of the chamber [0125] 105: Gap of the first chamber [0126]
106: (Center) axis of the first chamber [0127] 110: Second Chamber
[0128] 111: Top end of the second chamber [0129] 112: Bottom end of
the second chamber [0130] 113: First wall of the second chamber
[0131] 115: Gap of the second chamber [0132] 120: Third Chamber
[0133] 121: Top end of the third chamber [0134] 122: Bottom end of
the third chamber [0135] 123: First wall of the third chamber
[0136] 125: Gap of the third chamber [0137] 130: First thermal
brake [0138] 131: Top end of the first thermal brake [0139] 132:
Bottom end of the first thermal brake [0140] 133: First wall of the
first thermal brake, essentially contacting at least part of the
channel [0141] 140: Second thermal brake [0142] 141: Top end of the
second thermal brake [0143] 142: Bottom end of the second thermal
brake [0144] 143: First wall of the second thermal brake,
essentially contacting at least part of the channel [0145] 160:
Heating/cooling elements [0146] 160a: Heating (and/or cooling)
element of the first heat source [0147] 160b: Heating (and/or
cooling) element of the second heat source [0148] 170: Temperature
Sensors [0149] 170a: Temperature sensor of the first heat source
[0150] 170b: Temperature sensor of the second heat source [0151]
200: First securing element comprising at least one of following
elements [0152] 201: Screw or fastener (typically made of a thermal
insulator) [0153] 202a: Washer or positioning standoff (typically
made of a thermal insulator) [0154] 202b: Spacer or positioning
standoff (typically made of a thermal insulator) [0155] 203a:
Securing element of the first heat source [0156] 203b: Securing
element of the second heat source [0157] 210: Second securing
element (typically made as a wing structure) [0158] Used to
assemble the heat source assembly to the first housing element 300
[0159] 300: First housing element [0160] 310: Second insulator (or
second insulating gap) [0161] Located between the sides of the heat
sources and the side walls of the first housing element; and [0162]
Filled with a thermal insulator such as air, a gas, or a solid
insulator [0163] 320: Third insulator (or third insulating gap)
[0164] Located between the bottom of the first heat source and the
bottom wall of the first housing element; and [0165] Filled with a
thermal insulator such as air, a gas, or a solid insulator [0166]
330: Support [0167] 400: Second housing element [0168] 410: Fourth
insulator (or Fourth insulating gap) [0169] Located between the
side walls of the first housing element and those of the second
housing element; and [0170] Filled with a thermal insulator such as
air, a gas, or a solid insulator [0171] 420: Fifth insulator (or
fifth insulating gap) [0172] Located between the bottom wall of the
first housing element and that of the second housing element; and
[0173] Filled with a thermal insulator such as air, a gas, or a
solid insulator. [0174] 500: Centrifuge unit [0175] 501: Motor
[0176] 510: Axis of centrifugal rotation [0177] 520: Rotation arm
[0178] 530: Tilt shaft [0179] 600-603: Optical detection units
[0180] 610: Optical port [0181] 620: Light source [0182] 630:
Excitation lens [0183] 635: Lens [0184] 640: Excitation filter
[0185] 650: Detector [0186] 655: Aperture or slit [0187] 660:
Emission lens [0188] 670: Emission filter [0189] 680: Dichroic
beam-splitter [0190] 690: Reaction vessel cap [0191] 695: Optical
port [0192] 696: Bottom end of optical port [0193] 697: Top end of
optical port [0194] 698: Open space between inner wall of reaction
vessel and side wall of optical port [0195] 699: Side wall of
optical port
[0196] As discussed, and in one embodiment, the present invention
features a two-stage thermal convection apparatus adapted to
perform thermal convection PCR amplification.
[0197] In one embodiment, the apparatus includes as operably linked
components the following elements: [0198] (a) a first heat source
for heating or cooling a channel and comprising a top surface and a
bottom surface, the channel being adapted to receive a reaction
vessel for performing PCR, [0199] (b) a second heat source for
heating or cooling the channel and comprising a top surface and a
bottom surface, the bottom surface facing the top surface of the
first heat source, wherein the channel is defined by a bottom end
contacting the first heat source and a through hole contiguous with
the top surface of the second heat source, and further wherein
center points between the bottom end and the through hole form a
channel axis about which the channel is disposed, [0200] (c) at
least one temperature shaping element such as at least one gap or
space (e.g., a chamber) disposed around the channel and within at
least part of the second or first heat source, the chamber gap
being sufficient to reduce heat transfer between the second or
first heat source and the channel; and [0201] (e) a receptor hole
adapted to receive the channel within the first heat source.
[0202] In operation, the apparatus uses multiple heat sources such
as two, three, four or more heat sources, preferably two heat
sources positioned within the apparatus so that each is essentially
parallel to the other heat source in typical embodiments. In this
embodiment, the apparatus will generate a temperature distribution
suitable for a convection-based PCR process that is fast and
efficient. A typical apparatus includes a plurality of channels
disposed within the first and second heat sources so that a user
can perform multiple PCR reactions at the same time. For instance,
the apparatus can include at least one or two, three, four, five,
six, seven, eight, nine channels up to about ten, eleven, or twelve
channels, twenty, thirty, forty, fifty, or up to several hundred
channels extending through the first and second heat sources, with
between about eight to about one hundred channels being generally
preferred for many invention applications. A preferred channel
function is to receive a reaction vessel holding the user's PCR
reaction and to provide direct or indirect thermal communication
between the reaction vessel and at least one of and preferably all
of a) the heat sources, b) the temperature shaping element(s), and
c) the receptor hole.
[0203] The relative position of each of the two heat sources to the
other is an important feature of the invention. The first heat
source of the apparatus is typically located on the bottom and
maintained at a temperature suitable for nucleic acid denaturation,
and the second heat source is typically located on the top and
maintained at a temperature suitable for annealing of denatured
nucleic acid template with one or more oligonucleotide primers. In
some embodiments, the second heat source is maintained at a
temperature suitable for both annealing and polymerization. Thus in
one embodiment, the bottom part of the channel in the first heat
source and the top part of the channel in the second heat source
are subject to a temperature distribution suitable for the
denaturation and annealing steps of the PCR reaction, respectively.
In between the top and bottom part of the channel is the transition
region in which temperature change from the denaturation
temperature of the first heat source (the high temperature) to the
annealing temperature of the second heat source (the low
temperature) takes place. Thus, in typical embodiments, at least
part of the transition region is subject to a temperature
distribution suitable for polymerization of the primer along the
denaturated template. When the second heat source is maintained at
a temperature suitable for both annealing and polymerization, the
top part of the channel in the second heat source also provides a
temperature distribution suitable for the polymerization step in
addition to an upper part of the transition region. Therefore,
temperature distribution in the transition region is important for
achieving efficient PCR amplification, particularly regarding the
primer extension. Thermal convection inside the reaction vessel
typically depends on the magnitude and direction of the temperature
gradient generated in the transition region, and thus temperature
distribution in the transition region is also important for
generating suitable thermal convection inside the reaction vessel
that is conducive to PCR amplification. Various temperature shaping
elements can be used with the apparatus to generate a suitable
temperature distribution in the transition region to support fast
and efficient PCR amplification.
[0204] Typically, each individual heat source is maintained at a
temperature suitable for inducing each step of thermal convection
PCR. Moreover, and in embodiments in which the apparatus features
two heat sources, temperatures of the two heat sources are suitably
arranged to induce thermal convection across a sample inside a
reaction vessel. One general condition for inducing suitable
thermal convection according to the invention is, a heat source
maintained at a higher temperature is located at a lower position
within the apparatus than a heat source maintained at a lower
temperature. Thus in a preferred embodiment comprising two heat
sources, the first heat source is positioned lower in the apparatus
than the second heat source.
[0205] As discussed, it is an object of the invention to provide an
apparatus with at least one temperature shaping element. In most
embodiments, each channel of the apparatus will include less than
about ten of such elements, for example, one, two, three, four,
five, six, seven, eight, nine or ten of the temperature shaping
elements for each channel. One function of the temperature shaping
element is to provide for efficient thermal convection mediated PCR
by providing a structural or positional feature that supports PCR.
As will be more apparent from the examples and discussion which
follows, such features include, but are not limited to, at least
one gap or space such as a chamber; at least one insulator or
insulating gap located between the heat sources; at least one
thermal brake; at least one protrusion structure in at least one of
the first and second heat sources; at least one asymmetrically
disposed structure within the apparatus, particularly in at least
one of the channels, first heat source, second heat source, gap
such as a chamber, thermal brake, protrusion, first insulator, or
the receptor hole; or at least one structural or positional
asymmetry. Structural asymmetry is typically defined in reference
to the channel and/or channel axis. An example of positional
asymmetry is tilting or otherwise displacing the apparatus with
respect to the direction of gravity.
[0206] The words "gap" and "space" will often be used herein
interchangeably. A gap is a small enclosed or semi-enclosed space
within the apparatus that is intended to assist thermal convection
PCR. A large gap or large space with a defined structure will be
referred to herein as a "chamber". In many embodiments, the chamber
will include a gap and be referred to herein as a "chamber gap". A
gap may be empty, filled or partially filled with an insulating
material as described herein. For many applications, a gap or
chamber filled with air will be generally useful.
[0207] One or a combination of temperature shaping elements (the
same or different) can be used with the invention apparatus.
Illustrative temperature shaping elements will now be discussed in
more detail.
[0208] Illustrative Temperature Shaping Elements
[0209] A. Gap or Chamber
[0210] In one embodiment of the present apparatus, each channel
will include at least one gap or chamber as the temperature shaping
element. In a typical embodiment, the apparatus will include one,
two or even three chambers disposed around each channel and within
at least the second heat source. Alternatively, or in addition, the
apparatus may feature at least one chamber that is disposed around
the channel within the first heat source. However for many
embodiments, it is preferred to have at least one chamber disposed
around the channel within the second heat source, but no chamber
structure disposed within the first heat source. In this example of
the invention, the chamber creates a space between the channel and
the second (or sometimes first) heat source that allows the user to
precisely control temperature distribution within the apparatus.
That is, the chamber assists in shaping the temperature
distribution of the channel in the transition region. By
"transition region" is meant the region of the channel roughly in
between an upper part of the channel that contacts the second heat
source and a lower part of the channel that contacts the first heat
source. The chamber can be positioned nearly anywhere around the
channel provided intended results are achieved. For instance,
positioning the chamber (or more than one chamber) within or near
the second heat source will be useful in many invention
applications. Although less preferred, the chamber may also reside
in the first heat source or both the first and second heat sources.
In embodiments in which a channel in the apparatus has multiple
chambers, each chamber may be separated from the other and may in
some instances contact one or more other chambers within the
apparatus.
[0211] One or a combination of different gap or chamber structures
is compatible with the invention. As general requirements, the
chamber should generate a temperature distribution in the
transition region that fulfills at least one and preferably all of
the following conditions: (1) the temperature gradient generated
(particularly across the vertical profile of the channel) must be
large enough so as to generate a thermal convection across the
sample inside the reaction vessel; and (2) the thermal convection
thus generated by the temperature gradient must be sufficiently
slow (or appropriately fast) so that sufficient time periods can be
provided for each step of the PCR process. In particular, it is
especially important to make the time period of the polymerization
step sufficiently long since the polymerization step typically
takes more time than the denaturation and annealing steps. Examples
of particular gap or chamber configurations are disclosed
below.
[0212] If desired, the channel within an invention apparatus may
have at least one chamber disposed essentially symmetrically or
asymmetrically about the channel axis. In many embodiments, an
apparatus with one, two or three chambers will be preferred. The
chambers may be disposed in one or a combination of the heat
sources, for example, the second heat source, the first heat
source, or both the second and first heat sources. For many
apparatuses, having one, two, or three chambers within the second
heat source will be especially useful. Examples of such chamber
embodiments are provided below.
[0213] In one embodiment, the chamber will be further defined by
what is referred to herein as a "protrusion" from at least one of
the first heat source and the second heat source. In a particular
embodiment, the protrusion will extend from the second heat source
toward the first heat source in a direction generally parallel to
the channel axis. Other embodiments are possible such as including
a second protrusion extending away from the top surface of the
second heat source generally parallel to the channel axis.
Additional embodiments include an apparatus with a protrusion
extending from the first heat source toward the second heat source
generally parallel to the channel axis. Still further embodiments
include an apparatus with a second protrusion extending away from
the bottom surface of the first heat source also generally parallel
to the channel axis. In some embodiments, the apparatus may
comprise at least one protrusion that is tilted with respect to the
channel axis. In these examples of the invention, it is possible to
substantially reduce the volume of the first and/or second heat
sources as well as the heat transfer between the two heat sources
while lengthening chamber dimensions along the channel axis. These
features have been found to enhance thermal convection PCR
efficiency while reducing power consumption.
[0214] FIGS. 2A, 3A, 4A, 5A, 11A, 11B, 12A, 14A, 18A, and 20A
provide a few examples of acceptable chambers for use with the
invention. Other suitable chamber structures are disclosed
below.
[0215] B. Thermal Brake
[0216] Each channel within an invention apparatus may include one,
two, three or more thermal brakes, typically one or two thermal
brakes to control the temperature distribution within the
apparatus. In many embodiments, the thermal brake will be defined
by a top and bottom end and a wall that will be in optional thermal
contact with the channel. The thermal brake is typically disposed
adjacent or near a wall of the gap or chamber (if present). An
undesirable intrusion of a temperature profile from one heat source
to another (typically from the first heat source to the second heat
source) can be controlled and usually reduced by including the
thermal brake as a temperature shaping element. As will be
described in more detail below, it was found that thermal
convection PCR amplification efficiency is sensitive to the
position and thickness of the thermal brake. An acceptable thermal
brake may be disposed with respect to the channel either
symmetrically or asymmetrically.
[0217] One or more thermal brakes as described herein may be placed
in nearly any position around each channel of the apparatus
provided intended results are achieved. Thus in one embodiment, a
thermal brake can be positioned adjacent or near a chamber within
the second heat source to block or reduce undesired heat flow from
the first heat source and achieve suitable PCR amplification.
[0218] FIGS. 4B, 13B, 14B, 20C, 23B, 24B, 26B, and 27B provide a
few examples of suitable thermal brakes for use with the invention.
Other suitable thermal brakes are disclosed below.
[0219] C. Positional or Structural Asymmetry
[0220] It was found that thermal convection PCR was faster and more
efficient when an invention apparatus included at least one
positional or structural asymmetric element, for example, one, two,
three, four, five, or six of such elements for each channel. Such
elements can be placed around one or more channels up to the entire
apparatus. Without wishing to be bound by theory, it is believed
that presence of an asymmetric element within the apparatus
increases the buoyancy force in ways that make the amplification
process faster and more efficient. It has been found that by
introducing at least one positional or structural asymmetry within
the apparatus that can cause "horizontally asymmetric heating or
cooling" with respect to the channel axis or the direction of
gravity, it is possible to assist thermal convection PCR. Without
wishing to be bound by theory, it is believed that an apparatus
with at least one asymmetric element therein breaks apparatus
symmetry with regard to heating or cooling the channel and helps or
enhances generation of the buoyancy force so as to make the
amplification process faster and more efficient. By a "positional
asymmetric element" is meant that a structural element that makes
the channel axis or the apparatus tilted with respect to the
direction of gravity. By a "structural asymmetric element" is meant
that a structural element that is not symmetrically disposed within
the apparatus with respect to the channel and/or channel axis.
[0221] As discussed, it is necessary to generate a vertical
temperature gradient inside a sample fluid in order to generate
thermal convection (and also to fulfill the temperature
requirements for the PCR process). However, even in the presence of
a vertical temperature gradient, the buoyancy force that induces
the thermal convection may not be generated if isothermal contours
of the temperature distribution are flat (i.e., horizontal) with
respect to the direction of gravity (i.e., the vertical direction).
Within such a flat temperature distribution, the fluid does not
experience any buoyancy force since each part of the fluid has the
same temperature (and thus the same density) as other parts of the
fluid at the same height. In symmetric embodiments, all the
structural elements are symmetric with respect to the channel or
channel axis and the direction of gravity is aligned essentially
parallel to the channel or channel axis. In such symmetric
embodiments, isothermal contours of the temperature distribution
inside the channel or the reaction vessel often become nearly or
perfectly flat with respect to the gravitational field, and thus it
is often difficult to generate the thermal convection that is
sufficiently fast. Without wishing to be bound by theory, it is
believed that presence of certain perturbations that can induce a
fluctuation or instability in the temperature distribution often
helps or enhances generation of the buoyancy force and makes the
PCR amplification faster and more efficient. For instance, a small
vibration that typically exists in usual environment may disturb
the near or perfectly flat temperature distribution, or a small
structural defect in the apparatus may break the symmetry of the
channel/chamber structure or the reaction vessel structure so as to
disturb the near or perfectly flat temperature distribution. In
such a perturbed temperature distribution, the fluid can have
different temperature for at least part of the fluid as compared to
other part of the fluid at the same height, and thus the buoyancy
force can be readily generated due to such temperature fluctuation
or instability. Such natural or incidental perturbations are
usually important in generating the thermal convection in the
symmetric embodiments. When a positional or structural asymmetry is
present within the apparatus, the temperature distribution within
the channel or the reaction vessel can be controllably made uneven
at the same height (i.e., horizontally uneven or asymmetric). In
the presence of such horizontally asymmetric temperature
distribution, the buoyancy force can be readily and usually more
strongly generated so as to make the thermal convection PCR faster
and more efficient. Useful positional or structural asymmetric
elements cause "horizontally asymmetric heating or cooling" of the
channel with respect to the channel axis or the direction of
gravity.
[0222] Asymmetry can be introduced into an invention apparatus by
one or a combination of strategies. In one embodiment, it is
possible to make an invention apparatus with a positional asymmetry
imposed on the apparatus, for example, by tilting the apparatus or
the channel with respect to the direction of gravity. Nearly any of
the apparatus embodiments disclosed herein can be tilted by
incorporating a structure capable of offsetting the channel axis
with respect to the direction of gravity. An example of an
acceptable structure is a wedge or related inclined shape, or an
inclined or tilted channel. See FIGS. 11A-B for examples of this
invention embodiment.
[0223] In other embodiments, at least one of the following elements
can be asymmetrically disposed within the apparatus with respect to
the channel axis: a) the channel, b) a gap such as a chamber, c)
the receptor hole d) the first heat source, e) the second heat
source, f) the thermal brake; and g) the insulator. Thus in one
invention embodiment, the apparatus features a chamber as the
structural asymmetric element. In this invention example, the
apparatus may include one or more other structural asymmetric
elements such as the channel, receptor hole, thermal brake,
insulator, or one or more of the heat sources. In another
embodiment, the structural asymmetric element is the receptor hole.
In yet another embodiment, the structural asymmetric element is the
thermal brake or more than one thermal brake. The apparatus may
include one or more other asymmetric or symmetric structural
elements such as the first heat source, the second heat source, the
chamber, the channel, the insulator etc.
[0224] In embodiments in which the first heat source and/or the
second heat source feature a structural asymmetric element, the
asymmetry may reside particularly in a protrusion (or more than one
protrusion) that extends generally parallel to the channel
axis.
[0225] Further examples are provided below. In particular, see
FIGS. 17A-B, 18A-D, 19A-B, 21, and 22.
[0226] As discussed, one or both of the channel and chamber can be
symmetrically or asymmetrically disposed in the apparatus with
respect to the channel axis. See also FIGS. 8A-J, 9A-I, and 10A-P
for examples in which the channel and/or chamber are the symmetric
or asymmetric structural element.
[0227] It will often be desirable to have an apparatus in which the
receptor hole is the structural asymmetric element. Without wishing
to be bound to any theory, it is believed that the region between
the receptor hole and the bottom end of the chamber or the second
heat source is a location in the apparatus where a major driving
force for thermal convection flow is generated. As will be readily
apparent, this region is where initial heating to the highest
temperature (i.e., the denaturation temperature) and transition
toward a lower temperature (i.e., the polymerization temperature)
take place, and thus the largest driving force should originate
from this region.
[0228] See, for example, FIGS. 15 and 17A-B showing asymmetric
receptor hole structures.
[0229] D. Insulator and Insulating Gap
[0230] It will often be useful to insulate each of the heat sources
from the other to achieve the objects of this invention. As will be
apparent from the following discussion, the apparatus can be used
with a wide variety of insulators placed in the insulating gap
between the heat sources. Thus in one embodiment, a first insulator
is placed in the first insulating gap between the first and second
heat sources. One or a combination of gas or solid insulators
having low thermal conductivity can be used. A generally useful
insulator for many purposes of the invention is air (having low
thermal conductivity of about 0.024 Wm.sup.-1K.sup.-1 at room
temperature for static air, with a gradual increase with increasing
temperature). Although materials that have a thermal conductivity
larger than that of static air can be used without significantly
reducing the performance of the apparatus other than the power
consumption, it is generally preferred to use gas or solid
insulators that have a thermal conductivity similar to or smaller
than air. Examples of good thermal insulators include, but not
limited to, wood, cork, fabrics, plastics, ceramics, rubber,
silicon, silica, carbon, etc. Rigid foams made of such materials
are particularly useful since they represent very low thermal
conductivity. Examples of rigid foams includes, but not limited to,
Styrofoam, polyurethane foam, silica aerosol, carbon aerosol,
SEAgel, silicone or rubber foam, wood, cork, etc. In addition to
air, polyurethane foam, silica aerosol and carbon aerosol are
particularly useful thermal insulators to use at elevated
temperatures.
[0231] In embodiments in which an invention apparatus has the
insulating gap, advantages will be apparent. For instance, a user
of the apparatus will have the ability to 1) reduce the power
consumption by substantially reducing heat transfer from one heat
source to next heat source; and 2) control the temperature gradient
for generating the driving force (and therefore control the thermal
convection) since large temperature change from one heat source to
next heat source occurs in the insulating gap region. It has been
found that a larger insulating gap with a low thermal conductivity
insulator generally helps reducing the power consumption. Use of
the protrusion structures is particularly useful for substantially
reducing the power consumption since a larger average gap can be
provided while independently controlling different regions of the
insulating gap (i.e., regions near and distant from the channel,
separately). It has been also found that by changing the insulating
gap, particularly in the region near the channel, it is possible to
control the speed of the thermal convection and thus the speed of
the PCR amplification. Other advantages of having the insulating
gap will be apparent from the discussion and Examples that
follow.
[0232] It will be apparent from the following discussion and
examples that an invention apparatus may include one or a
combination of the foregoing temperature shaping elements. Thus in
one embodiment, the apparatus features at least one chamber (e.g.,
one, two or three chambers) disposed symmetrically about the
channel and typically parallel to the channel axis along with the
first insulator separating the first and second heat sources from
each other. In this embodiment, the apparatus may further include
one or two thermal brakes to further assist thermal convection PCR.
In an embodiment in which the apparatus includes two chambers, for
instance within the second heat source, each chamber may have the
same or different horizontal position with respect to the channel
axis. In another embodiment, the second heat source features a
first protrusion extending toward the first heat source; and
optionally a second protrusion extending away from the top surface
of the second heat source generally parallel to the channel axis,
in which the first protrusion typically defines the chamber. In
this embodiment, the apparatus may further include a first
protrusion extending from the first heat source to the second heat
source; and optionally a second protrusion extending away from the
bottom surface of the first heat source generally parallel to the
channel axis. In these embodiments, the second heat source
typically includes at least one chamber (e.g., one, two or three
chambers) disposed symmetrically with respect to the channel axis,
and the first heat source typically includes no chamber, but
sometimes may include one chamber or two chambers disposed
symmetrically with respect to the channel axis.
[0233] As discussed, it will often be useful to include asymmetric
structural element within the apparatus. Thus it is an object of
the invention to include within the apparatus a receptor hole that
is disposed asymmetrically with respect to the channel axis. In
this embodiment, the apparatus may include one or more chambers
disposed symmetrically or asymmetrically with respect to the
channel axis. Alternatively, or in addition, the apparatus may
feature at least one thermal brake that is disposed asymmetrically
with respect to the channel axis. In this embodiment, the apparatus
may include one or more chambers disposed symmetrically or
asymmetrically with respect to the channel axis. Alternatively, or
in addition, the apparatus may feature at least one of the
protrusions disposed asymmetrically with respect to the channel
axis. In one embodiment, the protrusion extending from the first
heat source is disposed asymmetrically about the channel axis while
one or both protrusions (and chamber) extending from the second
heat source is disposed symmetrically or asymmetrically about the
channel axis. Alternatively, or in addition, the one or more
protrusions (and chamber) of the second heat source can be disposed
asymmetrically about the channel axis while one or both protrusions
extending from the first heat source is disposed symmetrically or
asymmetrically about the channel axis.
[0234] However, in another embodiment, one or more of the channels
up to all of the channels within the apparatus need not include any
chamber or gap structure. In this example, the apparatus will
preferably include one or more other temperature shaping elements
such as tilting the angle of the channel with respect to gravity
(an example of positional asymmetry). Alternatively, or in
addition, the channel can include a structural asymmetry or be
subjected to centrifugal acceleration as provided herein.
[0235] As will be appreciated, it is possible to have an invention
apparatus in which other or further asymmetric elements are
present. For example, the apparatus can include two or three
chambers in which one or more of the chambers are disposed
asymmetrically with respect to the channel axis. In embodiments in
which the apparatus includes a single chamber, that chamber may be
disposed asymmetrically with respect to the channel axis.
Embodiments include an apparatus in which protrusions extending
from the second heat source toward the first heat source are
disposed asymmetrically with respect to the channel axis.
[0236] If desired, any of the foregoing invention embodiments can
include a positional asymmetry by tilting the device or the channel
with respect to the direction of gravity or placing it on a wedge
or other inclined shape.
[0237] As will be appreciated, nearly any temperature shaping
element of an apparatus embodiment (whether symmetrically or
asymmetrically disposed within the apparatus with respect to the
channel axis) can be combined with one or more other temperature
shaping elements including other structural or positional features
of the apparatus so long as intended results are achieved.
[0238] As will also be appreciated, the invention is flexible and
includes an apparatus in which each channel includes the same or
different temperature shaping elements. For example, one channel of
the apparatus can have no chamber or gap structures while another
channel of the apparatus includes one, two, or three of such
chamber or gap structures. The invention is not limited to any
channel configuration (or group of channel configurations) so long
as intended results are achieved. However, it will often be
preferred to have all the channels of an invention apparatus have
the same number and type of temperature shaping element to simplify
use and manufacturing considerations.
[0239] Reference to the following figures and examples is intended
to provide greater understanding of the thermal convection PCR
apparatus. It is not intended and should not be read as limiting
the scope of the present invention.
[0240] Turning now to FIGS. 1 and 2A-C, the apparatus 10 features
the following elements as operably linked components: [0241] (a) a
first heat source 20 for heating or cooling a channel 70 and
comprising a top surface 21 and a bottom surface 22 in which the
channel 70 is adapted to receive a reaction vessel 90 for
performing PCR; [0242] (b) a second heat source 30 for heating or
cooling the channel 70 and comprising a top surface 31 and a bottom
surface 32 in which the bottom surface 32 faces the top surface of
the first heat source 21, wherein the channel 70 is defined by a
bottom end 72 contacting the first heat source 20 and a through
hole 71 contiguous with the top surface of the second heat source
41. In this embodiment, center points between the bottom end 72 and
the through hole 71 form a channel axis 80 about which the channel
70 is disposed; [0243] (c) at least one chamber disposed around the
channel 70 and within at least part of the second heat source 30.
In this embodiment, the first chamber 100 includes a chamber gap
105 between the second heat source 30 and the channel 70 sufficient
to reduce heat transfer between the second heat source 30 and the
channel 70; and [0244] (d) a receptor hole 73 adapted to receive
the channel 70 within the first heat source 20.
[0245] By the phrase "operably linked", "operably associated" or
like phrase is meant one or more elements of the apparatus that are
operationally linked to one or more other elements. More
specifically, such an association can be direct or indirect (e.g.,
thermal), physical and/or functional. An apparatus in which some
elements are directly linked and others indirectly (e.g.,
thermally) linked is within the scope of the present invention.
[0246] In the embodiment shown in FIG. 2A, the apparatus further
includes a first insulator 50 positioned between the top surface 21
of the first heat source 20 and the bottom surface 32 of the second
heat source 30. As will be appreciated, practice of the invention
is not limited to having only one insulator present provided the
number of insulators is sufficient for intended results to be
achieved. That is, the invention may include multiple insulators
(e.g. 2, 3 or 4 insulators). In most embodiments, it is preferred
to have the length of the second heat source 30 that is greater
than the length of the first heat source 20 along the channel axis
80. Although in other embodiments the length of the second heat
source 30 can be smaller or essentially the same as that of the
first heat source 20, it is advantageous to have a greater length
for the second heat source 30 to achieve a longer path length for
the polymerization step.
[0247] In one embodiment shown in FIG. 2A, the first insulator 50
is filled with a thermal insulator having a low thermal
conductivity. Preferred thermal insulators have a thermal
conductivity between about a few tenths of Wm.sup.-1K.sup.-1 to
about 0.01 Wm.sup.-1K.sup.-1 or smaller. In this embodiment, the
length of the first insulator 50 along the channel axis 80 is made
to be small, for instance, between about 0.1 mm to about 5 mm,
preferably between about 0.2 mm to about 4 mm. In this example of
the invention, heat loss from one heat source to an adjacent heat
source can be substantially large, resulting in large power
consumption in operating the apparatus. For many applications, it
will often be preferred to have the two heat sources (e.g., 20 and
30) isolated from each other and also preferably isolated from
other elements of the apparatus if exist. Use of one or more
thermal insulators will often be helpful. For instance, use of a
thermal insulator in the first insulating gap 50 can often lower
power consumption.
[0248] Thus in the invention embodiment of the invention shown in
FIGS. 2A-C, the first insulator 50 comprises or consists of a solid
or a gas as a thermal insulator.
[0249] Turning again to the apparatus shown in FIGS. 2A-C, the
chamber gap 105 between the chamber wall 103 and the channel 70
inside the second heat source may be partially or totally filled
with a thermal insulator such as a gas, solid, or gas-solid
combination. Typically useful insulators include air, and gas or
solid insulators that have a thermal conductivity similar to or
smaller than air. Since one important function of the chamber gap
105 is to control (typically to reduce) heat transfer from the
second heat source to the channel inside the second heat source,
materials that have a thermal conductivity larger than that of air
such as plastics or ceramics can also be used. However, when such
higher thermal conductivity materials are used, the chamber gap 105
should be adjusted to be larger compared to the embodiment of using
air as an insulator. Similarly, if a material having a lower
thermal conductivity than air is used, the chamber gap 105 should
be adjusted to be smaller than that of the air insulator
embodiment.
[0250] In particular, FIGS. 2A-C show an apparatus embodiment in
which air or a gas is used as an insulator in the first insulator
50 and the chamber gap 105. The channel structures inside these
gaps are depicted with dashed lines to represent invisibility of
these structures when air (or a gas) is used as an insulator. If
desired to achieve a particular invention objective, the apparatus
can be adapted so that a solid insulator is used in the chamber gap
105. Alternatively, or in addition, the apparatus may include a
solid insulator in the first insulator 50.
[0251] FIGS. 2B and 2C show perspective views of section A-A and
B-B of the apparatus as marked in FIG. 1. An embodiment in which
air or a gas is used as an insulator is shown.
[0252] As shown in the embodiment of FIGS. 1 and 2A-C, the
apparatus features twelve channels (sometimes referred herein to as
reaction vessel channels). However, more or less channels are
possible depending on intended use, for instance, from about one or
two to about twelve channels, or between about twelve to several
hundred channels, preferably about eight to about one hundred
channels. Preferably, each channel is independently adapted to
receive a reaction vessel 90 that is typically defined by a bottom
end 92 within the first heat source 20 and a top end 91 on the top
of the second heat source 31. The channel 70 in the first 20 and
second 30 heat sources typically passes through the first insulator
50. Center points between the top 71 and bottom 72 ends of the
channel 70 form an axis of the channel 80 (sometimes referred
herein to as channel axis) about which the heat sources and
insulators are disposed.
[0253] Referring again to the embodiment shown in FIGS. 1 and 2A-C,
the channel 70 is adapted so that the reaction vessel 90 can fit
snugly therein i.e., it has a dimensional profile that is
essentially the same as that of a lower part of the reaction vessel
as depicted in FIG. 2A. In the operation, the channel functions as
a receptor for receiving a reaction vessel. However as will be
explained in more detail below, the structure of the channel 70 can
be adjusted and/or moved in relation to the channel axis 80 to
provide different thermal contact possibilities between the
reaction vessel 90 and one or more of the heat sources 20 and
30.
[0254] As an example, the through hole 71 formed in the second heat
source 30 can function as a top part of the channel 70. In this
embodiment, the channel 70 inside the second heat source 30 is in
physical contact with the second heat source 30. That is, a wall of
the through hole 71 extending into the second heat source 30 is in
physical contact with the reaction vessel 90. In this embodiment,
the apparatus can provide efficient heat transfer from the second
heat source 30 to the channel 70 and reaction vessel 90.
[0255] For many invention applications, it will be generally
preferred to have the size of the through hole in the second heat
source essentially the same as that of the channel or reaction
vessel. However, other through hole embodiments are within the
scope of the present invention and are disclosed herein. For
example, and referring again to FIGS. 2A-C, the through hole 71 in
the second heat source 30 may be made larger than the size of the
reaction vessel 90. However, in such case, heat transfer from the
second heat source 30 to the reaction vessel 90 may become less
efficient. In this embodiment, it may be useful to lower the
temperature of the second heat source 30 for optimal practice of
the invention. For most invention applications, it will be
generally useful to have the size of the through hole 71 in the
second heat source 30 essentially the same size as that of the
reaction vessel 90.
[0256] In invention embodiments in which the receptor hole 73 has a
closed bottom end 72 formed in the first heat source 20, it will
often function as a bottom portion of the channel 70. See FIG. 2A,
for instance. In such an embodiment, the receptor hole 73 of the
first heat source 20 has a size essentially the same as that of the
bottom part of the reaction vessel 92 which in most embodiments
will provide physical contact and efficient heat transfer to the
reaction vessel 90. In some invention embodiments, the receptor
hole 73 in the first heat source 20 may have a partial chamber
structure or a size slightly larger than that of the bottom part of
the reaction vessel as will be discussed.
[0257] Chamber Structure and Function
[0258] Turning again to the apparatus shown in FIGS. 2A-C, the
first chamber 100 is symmetrically disposed about the channel 70
and within the second heat source 30. Presence of such a physically
non-contacting (but thermally contacting) space within the
apparatus 10 provides many benefits and advantages. For example,
and without wishing to be bound to any theory, presence of the
first chamber 100 provides heat transfer from the second heat
source 30 to the channel 70 or the reaction vessel 90 that is
desirably less efficient. That is, the chamber 100 reduces heat
transfer substantially between the second heat source 30 and the
channel 70 or the reaction vessel 90. As will become more apparent
from the discussion that follows, this invention feature supports
robust and faster thermal convection PCR within the apparatus
10.
[0259] While it will often be useful to include a physically
non-contacting space within the second heat source 30, it is within
the scope of the present invention to include such a space within
the first heat source 20. For example, the first heat source 20 may
include one or more chambers intended to reduce heat transfer
between the first heat source 20 and the channel 70 or the reaction
vessel 90.
[0260] The invention embodiment shown in FIGS. 2A-C includes a
first chamber 100 in the second heat source 20 as a key structural
element. In this example of the invention, the first chamber 100 is
independently adapted to receive the channel 70 from the top of the
second heat source 31 toward the bottom of the second heat source
32 and the top of the first heat source 21. The first chamber 100
is defined by a top end 101 within the second heat source 30, a
bottom end 102 on the bottom of the second heat source 30, and the
first chamber wall 103 that is disposed around the channel axis 80
and spaced from the channel 70 inside the second heat source 30.
The chamber wall 103 surrounds the channel 70 inside the second
heat source 20 at a distance, forming a chamber gap 105. The
chamber gap 105 between the chamber wall 103 and the channel 70 is
preferably in the range between from about 0.1 mm to about 6 mm,
more preferably from about 0.2 mm to about 4 mm. The length of the
first chamber 100 is between about 1 mm to about 25 mm, preferably
between about 2 mm to about 15 mm.
[0261] The invention is compatible with a wide variety of heat
source and insulator configurations. For instance, the first heat
source 20 can have a length larger than about 1 mm along the
channel axis 80, preferably from about 2 mm to about 10 mm; and the
second heat source 30 can have a length between from about 2 mm to
about 25 mm along the channel axis 80, preferably from about 3 mm
to about 15 mm. As discussed, it will be generally useful to have
an apparatus with a first insulator 50. For example, in embodiments
without the protrusions, the first insulator 50 can have a length
along the channel axis 80 between about 0.2 mm to about 8 mm along
the channel axis 80, preferably between about 0.5 mm to 5 mm. In
other embodiments in which the protrusion structure is present, the
first insulator 50 can have different lengths along the channel
axis 80 depending on the position with respect to the channel 70.
For instance, in the region near or around the channel (i.e.,
within the protrusions), the first insulator 50 can have a length
along the channel axis between about 0.2 mm to about 8 mm,
preferably between about 0.5 mm to 5 mm. In the region distant from
the channel (i.e., outside the protrusion structures), the first
insulator 50 can have a length along the channel axis between about
0.5 mm to about 20 mm, preferably between about 1 mm to 10 mm.
[0262] As discussed, an invention apparatus may include multiple
chambers (for example, two, three, four or more chambers) within at
least one of the heat sources such as the second heat source.
[0263] In the embodiment shown in FIGS. 3A-B, the apparatus
includes a first chamber 100 positioned entirely within the second
heat source 30. In this embodiment, the first chamber 100 includes
the chamber top end 101 facing a first chamber bottom end 102 along
the channel axis 80. The apparatus further includes a second
chamber 110 positioned entirely within the second heat source 30
and in contact with the top end 101 of the first chamber 100. The
wall 103 of the first chamber 100 is aligned essentially parallel
to the channel axis 80. The second chamber 110 is further defined
by the wall 113 positioned essentially parallel to the channel axis
80. The second chamber 110 is further defined by a top end 111
within the second heat source 30 and a bottom end 112 in contact
with the top end 101 of the first chamber 100. As shown, the first
chamber 100 and the second chamber 110 include gaps 105 and 115,
respectively. In the embodiment shown, each of the top end 111 and
bottom end 112 of the second chamber 110 are perpendicular to the
channel axis 80. As shown in FIG. 3A, the width or radius of the
first chamber 100 from the channel axis 80 is smaller (about 0.9 to
0.3 times smaller) than the width or radius of the second chamber
110 from the channel axis 80. However as shown in the embodiment of
FIG. 3B, the width or radius of the first chamber 100 from the
channel axis 80 is greater (about 1.1 to about 3 times greater)
than the width of the second chamber 110 from the channel axis
80.
[0264] Turning again to FIGS. 3A-B, the first chamber 100 and the
second chamber 110 provide a useful temperature controlling or
shaping effect. In these embodiments, the first chamber 100 (FIG.
3A) or the second chamber 110 (FIG. 3B) has a smaller diameter or
width compared to the other chamber. The narrower portion of the
second chamber 110 (FIG. 3B) or first chamber 100 (FIG. 3A)
provides more efficient heat transfer from the second heat source
30 compared to the other chamber. In addition, the chamber
configuration shown in these embodiments blocks or reduces heat
transfer from the first heat source.
[0265] Unless otherwise mentioned, embodiments with multiple
chambers will be described by numbering the chambers from the first
heat source (typically located nearest the bottom of the
apparatus). Thus the chamber closest to the first heat source will
be designated "first chamber", the next closest chamber to the
first heat source will be designated "second chamber", etc.
[0266] Thermal Brake Structure and Function FIG. 4A shows an
invention embodiment with two chambers positioned in the second
heat source. In particular, the apparatus 10 has the first chamber
100 and the second chamber 110 positioned in the second heat source
30.
[0267] FIG. 4B is an expanded view of the dotted circle shown in
FIG. 4A. In particular, the region between the first chamber 100
and the second chamber 110 defines a first thermal brake 130. As
mentioned above, the first thermal brake 130 is intended to control
the temperature distribution within the apparatus 10. In the
embodiment shown, the first thermal brake 130 is defined by a top
end 131 and a bottom end 132 and a wall 133 that essentially
contacts the channel 70. In this embodiment, a function of the
first thermal brake 130 is to reduce or block an undesirable
intrusion of a temperature profile from the first heat source 20 to
the second heat source 30. Another function of the first thermal
brake 130 is to provide an efficient heat transfer between the
second heat source 30 and the channel 70 so as to make the channel
in that region quickly approach the temperature of the second heat
source 30. The first thermal brake 130 is disposed symmetrically
about the channel 70.
[0268] If desired, at least one of the first chamber 100 and the
second chamber 110 (or a portion thereof) may include a suitable
solid or a gas insulator. Alternatively, or in addition, the first
insulator 50 shown may include or consist of a suitable solid or a
gas. An example of suitable insulating gas is air.
[0269] Protrusion Structure and Function
[0270] In many invention embodiments, the apparatus 10 features at
least one protrusion extending from the top or bottom surface of
the first or second heat source. In one embodiment, the second heat
source 30 features a first protrusion 33 extending from the bottom
surface 32 of the second heat source 30 toward the first heat
source 20 in a direction generally parallel to the channel axis;
and optionally a second protrusion 34 extending away from the top
surface 31 of the second heat source 30 generally parallel to the
channel axis. Alternatively, or in addition, the first heat source
20 may include a first protrusion 23 extending from the top surface
21 of the first heat source 20 toward the second heat source 30
generally parallel to the channel axis; and optionally a second
protrusion 24 extending away from the bottom surface 22 of the
first heat source 20 generally parallel to the channel axis. In
some embodiments, the apparatus may comprise at least one
protrusion that is tilted with respect to the channel axis.
[0271] FIGS. 5A-C show an invention embodiment comprising a first
protrusion 33 of the second heat source 30 extending toward the
first heat source 20 and a first protrusion 23 of the first heat
source 20 extending toward the second heat source 30. In this
example of the invention, each of the protrusions (23, 33) is
disposed symmetrically about the first chamber 100 and/or the
channel axis 80. In this embodiment, the first protrusion 33 of the
second heat source 30 helps define the first chamber 100 or the
channel 70, the first insulator 50, and the second heat source 30,
and separate the first insulator 50 from the first chamber 100 or
the channel 70. The first protrusion 23 of the first heat source 20
helps define the channel 80 and the first heat source 20, and
separate the first insulator 50 from the channel 70. The
protrusions 23, 33 also define a portion 51 of the first insulator
50 (called a first insulator chamber). In this embodiment, the
first insulator chamber 51 is defined by at least the first heat
source 20, the first protrusion of the first heat source 23, the
second heat source 30, and the first protrusion of the second heat
source 33.
[0272] In the embodiment shown in FIGS. 5A-C, the top 101 and
bottom 102 ends of the first chamber 100 are essentially
perpendicular to the channel axis 80. The length of the first
chamber 100 is between about 1 mm to about 25 mm, preferably
between about 2 mm to about 15 mm. Additionally, the receptor hole
73 is symmetrically disposed about the channel 70 and channel axis
80.
[0273] In this embodiment, the function of the protrusions 23 and
33 is to reduce the heat transfer between the first 20 and second
30 heat sources as well as the volume of the first 20 and second 30
heat sources while lengthening the chamber dimension along the
channel axis to assist the thermal convection PCR. By use of the
protrusion structures, the first insulating gap can be made small
near the channel region (i.e., within the protrusions structures)
so that a longer chamber length along the channel axis can be
provided to enhance the efficiency of the thermal convection PCR,
while providing a larger gap outside the protrusion structures to
help reduce the heat transfer between the two heat sources so as to
reduce the power consumption of the apparatus. The volume of the
two heat sources can also be reduced substantially by use of the
protrusion structures 23, 33 so that the heat capacity of the two
heat sources is reduced to further assist reduction of the power
consumption.
[0274] Referring to the embodiment shown in FIGS. 6A-C, the first
heat source 20 further includes a second protrusion 24 extending
away from the bottom surface 22 of the first heat source 20 in
addition to the first protrusion 23. The second heat source 30 also
further includes a second protrusion 34 extending away from the top
surface 31 of the second heat source in addition to the first
protrusion 33. Other features of this embodiment are the same as
the embodiment shown in FIGS. 5A-C. In this embodiment, the
function of the second protrusions 24 and 34 is to further reduce
the volume of the first and second heat sources so as to further
reduce the power consumption of the apparatus. The second
protrusions 24, 34 of the first and second heat sources are also
useful in this embodiment to assist fast cooling of the two heat
sources after completion of the thermal convection PCR using a
cooling element such as a fan.
[0275] Channel Structure
[0276] A. Vertical Profiles
[0277] The invention is fully compatible with several channel
configurations. For example, FIGS. 7A-D show vertical sections of
suitable channel configurations. As shown, the vertical profile of
the channel may be shaped as a linear (FIGS. 7C-D) or tapered (FIG.
7A-B) channel. In a tapered embodiment, the channel may be tapered
either from the top to the bottom or from the bottom to the top.
Although various modifications are possible regarding the vertical
profile of the channel (e.g., a channel having a side wall that is
curved, or tapered with two or more different angles, etc.), it is
generally preferred to use a channel that is (linearly) tapered
from the top to the bottom because such structure facilitates not
only the fabrication process but also introduction of the reaction
vessel to the channel. A generally useful taper angle (.theta.) is
in the range between from about 0.degree. to about 15.degree.,
preferably from about 2.degree. to about 10.degree..
[0278] In the embodiments shown in FIGS. 7A-B, the channel 70 is
further defined by an open top 71 and a closed bottom end 72 which
ends may be perpendicular to the channel axis 80 (FIG. 7A) or
curved (FIG. 7B). The bottom end 72 may be curved with a convex or
concave shape having a radius of curvature equal to or larger than
the radius or half width of the horizontal profile of the bottom
end. Flat or near flat bottom end with its radius of curvature at
least two times larger than the radius or half width of the
horizontal profile of the bottom end is more preferred over other
shapes since it can provide an enhanced heat transfer for the
denaturation process. The channel 70 is further defined by a height
(h) along the channel axis 80 and a width (w1) perpendicular to the
channel axis 80.
[0279] For many invention applications, it will be useful to have a
channel 70 that is essentially straight (i.e., not bent or
tapered). In the embodiments shown in FIGS. 7C-D, the channel 70
has the open top end 71 and the closed bottom end 72 which may be
perpendicular to the channel axis 80 (FIG. 7C) or curved (FIG. 7D).
As in the tapered channel embodiments, the bottom end 72 may be
curved with a convex or concave shape and flat or near flat bottom
end having a large curvature is typically more preferred. The
channel 70 is further defined in these embodiments by a height (h)
along the channel axis 80 and a width (w1) perpendicular to the
channel axis 80.
[0280] In the channel embodiments shown in FIGS. 7A-D, the height
(h) is at least about 5 mm to about 25 mm, preferably 8 mm to about
16 mm for a sample volume of about 20 microliters. Each channel
embodiment is further defined by the average of the width (w1)
along the channel axis 80 which is typically at least about 1 mm to
about 5 mm. Each of the channel embodiments shown in FIGS. 7A-D can
be further defined by a vertical aspect ratio which is the ratio of
the height (h) to the width (w1), and a horizontal aspect ratio
which is the ratio of the first width (w1) to the second width (w2)
along first and second directions, respectively, that are mutually
perpendicular to each other and aligned perpendicular to the
channel axis. A generally suitable vertical aspect ratio is between
about 4 to about 15, preferably from about 5 to about 10. The
horizontal aspect ratio is typically between about 1 to about 4. In
embodiments in which the channel 70 is tapered (FIGS. 7A-B), the
width or diameter of the channel changes across the vertical
profile of the channel. By way of general guidance, for sample
volumes larger or smaller than 20 microliters, the height and width
(or diameter) may be scaled by a factor of cubic root or sometimes
square root of the volume ratio.
[0281] As discussed, the bottom end 72 of the channel may be flat,
rounded, or curved as depicted in FIG. 7A-D. When the bottom end is
rounded or curved, it typically has a convex or concave shape. As
discussed, a flat or near flat bottom end is more preferred over
other shapes for many invention embodiments. While not wishing to
be bound to any theory, it is believed that such a bottom design
can enhance heat transfer from the first heat source 20 to the
bottom end 71 of the channel 70 so as to facilitate the
denaturation process.
[0282] None of the foregoing vertical channel profiles are mutually
exclusive. That is, a channel that has a first portion that is
straight and second portion that is tapered (with respect to the
channel axis 80) is within the scope of the present invention.
[0283] B. Horizontal Profiles
[0284] The invention is also compatible with a variety of
horizontal channel profiles. An essentially symmetrical channel
shape is generally preferred where ease of manufacture is a
concern. FIGS. 8A-J show a few examples of acceptable horizontal
channel profiles, each with a designated symmetry. For instance,
the channel 70 may have its horizontal shape that is circular (FIG.
8A), square (FIG. 8D), rounded square (FIG. 8G) or hexagonal (FIG.
8J) with respect to the channel axis 80. In other embodiments, the
channel 70 may have a horizontal shape that has its width larger
than its length (or vice versa). For instance, and as depicted in
the middle column of FIGS. 8B, E and H, the horizontal profile of
the channel 70 may be shaped as an ellipsoid (FIG. 8B), rectangular
(FIG. 8E), or rounded rectangular (FIG. 8H). This type of
horizontal shape is useful when incorporating a convection flow
pattern going upward on one side (e.g., on the left hand side) and
going downward on the opposite side (e.g., on the right hand side).
Due to the relatively larger width profile incorporated compared to
the length, interference between the upward and downward convection
flows can be reduced, leading to more smooth circulative flow. The
channel may have a horizontal shape that has its one side narrower
than the opposite side. A few examples are shown on the right
column of FIGS. 8C, F and I. The left side of the channel is
depicted to be narrower than the right side for instance. This type
of horizontal shape is also useful when incorporating a convection
flow pattern going upward on one side (e.g., on the left hand side)
and going downward on the opposite side (e.g., on the right hand
side). Moreover, when this type of shape is incorporated, speed of
the downward flow (e.g., on the right hand side) can be controlled
(typically reduced) with respect to the upward flow. Since the
convective flow must be continuous within the continuous medium of
the sample, the flow speed should be reduced when cross-sectional
area becomes larger (or vice versa). This feature is particularly
important with regard to enhancing the polymerization efficiency.
The polymerization step typically takes place during the downward
flow (i.e., after the annealing step), and therefore time period
for the polymerization step can be lengthened by making the
downward flow slower as compared to that of the upward flow,
leading to more efficient PCR amplification.
[0285] Thus in one invention embodiment, at least part of the
channel 70 (including the entire channel) has a horizontal shape
along a plane essentially perpendicular to the channel axis 80. In
one invention example, the horizontal shape has at least one
reflection (.sigma.) or rotation symmetry element (C.sub.x) in
which X is 1, 2, 3, 4, up to .infin. (infinity). Nearly any
horizontal shape is acceptable provided it satisfies intended
invention objectives. Further acceptable horizontal shapes include
a circular, rhombus, square, rounded square, ellipsoid, rhomboid,
rectangular, rounded rectangular, oval, semi-circular, trapezoid,
or rounded trapezoid shape along the plane. If desired, the plane
perpendicular to the channel axis 80 can be within the first 20 or
second 30 heat source.
[0286] None of the foregoing horizontal channel profiles are
mutually exclusive. That is, a channel that has a first portion
that is circular, for instance, and a second portion that is
semi-circular (with respect to the channel axis 80) is within the
scope of the present invention.
[0287] Horizontal Chamber Shape and Position
[0288] As discussed, an apparatus of the invention can include at
least one chamber, preferably one, two or three chambers to help
control the temperature distribution within the apparatus, for
instance, within the transition region of the channel. The channel
can have one or a combination of suitable shapes provided intended
invention results are achieved.
[0289] For instance, FIGS. 9A-I show suitable horizontal profiles
of a chamber (the first chamber 100 is used as an illustration
only). In this invention embodiment, the horizontal profile of the
chamber 100 may be made into various different shapes although
shapes that are essentially symmetric will often be useful to
facilitate the fabrication process. For instance, the first chamber
100 may have a horizontal shape that is circular, square, or
rounded square as depicted in the left column. See FIGS. 9A, D, and
G. The first chamber 100 may have a horizontal shape that has its
width larger than its length (or vice versa), for instance, an
ellipsoid, rectangular, or rounded rectangular as depicted in the
middle column. The first chamber 100 may have a horizontal shape
that has its one side narrower than the opposite side as depicted
in the right column. See FIGS. 9C, F, and I.
[0290] As discussed, chamber structure is useful in controlling
(typically reducing) the heat transfer from the heat source
(typically the second heat source) to the channel or the reaction
vessel. Therefore, it is important to change the position of the
first chamber 100 relative to that of the channel 70 depending on
the invention embodiment of interest. In one embodiment, the first
chamber 100 is disposed symmetrically with respect to the position
of the channel 70, i.e., the chamber axis (an axis formed by the
center points of the top and bottom end of the chamber, 106)
coincides with the channel axis 80. In this embodiment, the heat
transfer from the heat source 20 or 30 to the channel is intended
to be constant in all directions across the horizontal profile of
the channel (at certain vertical location). Therefore, it is
preferred to use a horizontal shape of the first chamber 100 that
is the same as that of the channel in such embodiments. See FIGS.
9A-I.
[0291] However other embodiments of the chamber structure are
within the scope of the present invention. For instance, one or
more of the chambers within the apparatus may be disposed
asymmetrically with respect to the position of the channel 70. That
is the chamber axis 106 formed between the top end and bottom end
of a particular chamber may be off-centered, tilted or both
off-centered and tilted with respect to the channel axis 80. In
this embodiment, one or more of the chamber gaps between the
channel 70 and a wall of the chamber will be larger on one side and
smaller on the opposite side of that chamber. Heat transfer in such
embodiments will be higher in one side of the channel 70 and lower
in the opposite side (while it is same or similar in the two
opposite sides located along the direction perpendicular to the
positions of above two sides). In a particular embodiment, it is
preferred to use a horizontal shape of the first chamber 100 that
is circular or rounded rectangular. A circular shape is generally
more preferred.
[0292] Thus in one embodiment of the apparatus, at least part of
the first chamber 100 (including the entire chamber) has a
horizontal shape along a plane essentially perpendicular to the
channel axis 80. See FIG. 9A and FIG. 2A-C, for instance.
Typically, the horizontal shape has at least one reflection or
rotation symmetry element. Preferred horizontal shapes for use with
the invention include those that are circular, rhombus, square,
rounded square, ellipsoid, rhomboid, rectangular, rounded
rectangular, oval, semi-circular, trapezoid, or rounded trapezoid
shape along a plane perpendicular to the channel axis 80. In one
embodiment, the plane perpendicular to the channel axis 80 is
within the second 30 or first 20 heat source.
[0293] It will be appreciated that the foregoing discussion about
chamber structure and position will be applicable to more chamber
embodiments than the first chamber 100. That is, in an invention
embodiment with multiple chambers (e.g., one with the second
chamber 110 and/or third chamber 120), these considerations may
also apply.
[0294] Asymmetric and Symmetric Channel/Chamber Configurations
[0295] As mentioned, the invention is compatible with a wide
variety of channel and chamber configurations. In one embodiment, a
suitable channel is disposed asymmetrically with respect to the
chamber. FIGS. 10A-P show some examples of this concept.
[0296] In particular, FIGS. 10A-P show horizontal sections of
suitable channel and chamber structures with reference to location
of the channel 70 within the chamber 100 (the first chamber 100 is
used only for illustrative purposes). Horizontal shapes of the
first chamber 100 and channel 70 are shown to be circular or
rounded rectangular for instance. The first column (FIGS. 10A, E, I
and M) shows examples of symmetrically positioned structures. In
these embodiments, the chamber axis coincides with the channel axis
70. Therefore, the gap between the first chamber wall (103, solid
line) and the channel 70 (dotted line) is the same for the left and
right sides, and also for the upper and lower sides, providing a
heat transfer from the heat source to the channel that is symmetric
in both directions. The second column (FIGS. 10B, F, J and N) shows
examples of asymmetrically positioned structures. The channel axis
80 is positioned off-centered (to the left hand side) from the
chamber axis and the gap between the first chamber wall 103 and the
channel 70 is smaller on the left side (while it is the same on the
upper and lower sides), providing higher heat transfer from the
left side. The third (FIGS. 10C, G, K and O) and fourth (FIGS. 8D,
H, L, and P) columns show other examples of asymmetrically
positioned structures that provide more asymmetric heat transfer.
The third column (FIGS. 10C, G, K and O) shows examples in which
the chamber wall is in contact with the channel on one side (the
left side). The fourth column (FIGS. 10D, H, L, and P) shows
examples in which one side (the right side) forms the first chamber
100 while the opposite side (the left side) forms the channel 70.
In both examples, heat transfer from the left side is much higher
than from the right side. The physically contacting side shown in
the third and fourth columns is intended to function as a thermal
brake, particularly as an asymmetric thermal brake that provides
thermal braking on one side only.
[0297] It is thus an object of the invention to provide an
apparatus in which at least one of the chambers therein (e.g., one
or more of the first chamber 100, second chamber 110, or the third
chamber 120) is disposed essentially symmetrically about the
channel along a plane that is essentially perpendicular to the
channel axis. It is also an object to provide an apparatus in which
at least one of the chambers is disposed asymmetrically about the
channel and along the plane that is essentially perpendicular to
the channel axis. All or part of a particular chamber(s) can be
disposed about the channel axis either symmetrically or
asymmetrically as needed. In embodiments in which at least one
chamber is disposed asymmetrically about the channel axis, the
chamber axis and the channel axis can be off-centered while
essentially parallel to each other, tilted or both off-centered and
tilted. In a more specific embodiment of the foregoing, at least
part of a chamber including the entire chamber is disposed
asymmetrically about the channel along a plane perpendicular to the
channel axis. In other embodiments, at least part of the channel is
located inside the chamber along the plane perpendicular to the
channel axis. In one example of this embodiment, at least part of
the channel is in contact with the chamber wall along the plane
perpendicular to the channel axis. In another embodiment, at least
part of the channel is located outside of the chamber along the
plane perpendicular to the channel axis and contacting the second
or first heat source. For some invention embodiments, the plane
perpendicular to the channel axis contacts the second or first heat
source.
[0298] Vertical Chamber Shape
[0299] It is also an object of the invention to provide an
apparatus in which the second heat source includes at least one
chamber, typically one, two or three of same to help control
temperature distribution. Preferably, the chamber helps control the
temperature gradient of the transition region from one heat source
(e.g., the first heat source 20) within the apparatus to another
heat source (e.g., the second heat source 30) therein. Various
adaptations of the chamber are within the scope of the invention so
long as it generates a temperature distribution suitable for the
convection-based PCR process of the present invention.
[0300] It is an object of the invention to provide an apparatus in
which at least part of a chamber (up to and including the entire
chamber) is tapered along the channel axis. For instance, and in
one embodiment, one or more of the chambers including all of the
chambers therein are tapered along the channel axis. In one
embodiment, at least part of one or all of the chambers is
positioned within the second heat source and has a width (w)
perpendicular to the channel axis that is greater towards the first
heat source than the other side. In some embodiments, at least part
of the chamber is positioned within the second heat source and has
a width (w) perpendicular to the channel axis that is smaller
towards the first heat source than the other side. In one
embodiment, the apparatus includes the first chamber and the second
chamber positioned within the second heat source, the first chamber
having a width (w) perpendicular to the channel axis that is larger
(or smaller) than the width (w) of the second chamber. For some
embodiments, the first chamber is facing the first heat source.
[0301] Further Illustrative Apparatus Embodiments
[0302] Suitable heat source, insulator, channel, gap, chamber,
receptor hole configurations and PCR conditions are described
throughout the present application and may be used as needed with
the following invention examples.
[0303] A. One Chamber, First and Second Heat Sources,
Protrusion
[0304] In some invention embodiments, it will be useful to
manipulate the structure of one or more of the chambers by changing
the structure of at least one of the heat sources. For instance, at
least one of the first and second heat sources can be adapted to
include one or more protrusions that defines the gap or chamber and
generally extends essentially parallel to the channel or chamber
axis. A protrusion may be disposed symmetrically or asymmetrically
about the channel or chamber axis. Significant protrusions extend
away from one heat source to another heat source within the
apparatus. For example, the first protrusion of the second heat
source extends away from the second heat source in the direction
toward the first heat source and the first protrusion of the first
heat source extends away from the first heat source toward the
second heat source. In these embodiments, the protrusion contacts
the chamber and defines a chamber gap or chamber wall. In a
particular embodiment, the width or diameter of the second heat
source protrusion along the channel axis is decreased as going away
from the second heat source while the width of the first insulator
adjacent to the protrusion along the channel axis is increased.
Each chamber may have the same or different protrusion (including
no protrusion). An important advantage of the protrusions is to
help reduce the size of the heat sources and lengthen chamber
dimensions and insulator or insulating gap dimensions along the
channel axis. These and other benefits were found to assist thermal
convection PCR in the apparatus while substantially reducing the
power consumption of the apparatus.
[0305] A particular embodiment of an invention apparatus with
protrusions is shown in FIG. 5A. The apparatus includes a first
protrusion 33 of the second heat source 30 disposed essentially
symmetrically about the channel axis 80 and extending toward the
first heat source 20. The first chamber 100 is disposed within the
second heat source 30 and comprises a chamber wall 103 that is
essentially parallel to the channel axis 80. Importantly, there is
a gap between the bottom of the second heat source 32 and the top
of the first heat source 21. In this embodiment, the first heat
source 20 also includes a first protrusion 23 that are disposed
symmetrically about the channel 70 and extending toward the second
heat source 30. Also in this embodiment, the width or diameter of
the first heat source protrusions 23, 24 along the channel axis 80
is reduced as going away from the first heat source 20.
[0306] As is also shown in FIG. 5A, the receptor hole 73 is
disposed symmetrically about the channel axis 80. In this
embodiment, the receptor hole 73 has a width or diameter
perpendicular to the channel axis 80 that is about the same as the
width or diameter of the channel 70. Alternatively, the receptor
hole 73 may have a width or diameter perpendicular to the channel
axis 80 that is somewhat larger (for example, about 0.01 mm to
about 0.2 mm larger) than the width or diameter of the channel
70.
[0307] As discussed, it is an object of the invention to provide an
apparatus for performing thermal convection PCR which includes at
least one temperature shaping element which in one embodiment can
be a positional asymmetry imposed on the apparatus. FIG. 11A shows
one important example of this embodiment. As shown, the apparatus
is tilted at an angle .theta.g (tilting angle) with respect to the
direction of gravity. This type of embodiments is particularly
useful in controlling (typically increasing) speed of the thermal
convection PCR. Alternatively, the apparatus can be made to include
one or more of the channel and chambers that is tilted with respect
to the direction of gravity. FIG. 11B shows one example of such
embodiments in which both the channel and the first chamber are
tilted with respect to the direction of gravity. As will be
discussed below, increase of the tilting angle typically leads to
faster and more robust thermal convection PCR. Other embodiments
that include one or more positional asymmetries will be described
in more detail below.
[0308] The embodiments shown in FIGS. 5A and 11A will be
particularly suitable for many invention applications including
amplification of "difficult" samples such as genomic or chromosomal
target sequences or long-sequence target templates (e.g., longer
than about 1.5 or 2 kbp). In particular, FIG. 5A shows heat sources
with a symmetric chamber and channel configuration. The first
chamber 100 and the first protrusion 33 of the second heat source
30 effectively block protrusion of the high temperature of the
first heat source 20 toward inside the first chamber 100 as they
are located on the bottom of the second heat source 32. In use, the
temperature drops down rapidly in the first insulator region 50
from the high denaturation temperature (about 92.degree. C. to
about 106.degree. C.) of the first heat source 20 to the
polymerization temperature (about 80.degree. C. to about 60.degree.
C.) on the bottom part of the first chamber 100. Hence, the
temperature inside the first chamber 100 becomes more narrowly
distributed around the polymerization temperature (due to the early
cut off of the high denaturation temperature by the first thermal
brake) so that a large volume (and time) inside the second heat
source 30 becomes available for the polymerization step.
[0309] A major difference between the embodiments shown in FIGS. 5A
and 11A is that the apparatus of FIG. 11A has a tilting angle
.theta.g. The apparatus without the tilting angle (FIG. 5A) works
well and takes about 15 to 25 min to amplify from a 1 ng plasmid
sample and about 25 to 30 min to amplify from a 10 ng human genome
sample (3,000 copies) when the structure of the apparatus is
optimized. PCR amplification efficiency of the apparatus can be
further enhanced if a tilting angle of about 2.degree. to about
60.degree. (more preferably about 5.degree. to about 30.degree.) is
introduced as depicted in FIG. 11A. With the gravity tilting angle
introduced with this structure (FIG. 11A), PCR amplification from a
10 ng human genome sample can be completed in about 20 to 25 min.
See Examples 1 and 2 below.
[0310] B. Tapered Chamber
[0311] Referring now to FIGS. 12A-B, the apparatus embodiment
features a first chamber 100 that is concentric with the channel.
In this example of the invention, the chamber axis (i.e., an axis
formed by the centers of the top and bottom end of the chamber)
coincides with the channel axis 80. The chamber wall 103 of the
first chamber 100 has an angle with respect to the channel axis 80.
That is, the chamber wall 103 is tapered from the top end 101 to
the bottom end 102 of the first chamber 100 (FIG. 12A). In FIG.
12B, the chamber wall 103 is tapered from the bottom end 102 to the
top end 101 of the first chamber 100. Such a structure provides a
narrow hole on the bottom and a wide hole on the top, or vice
versa. For instance, if the bottom part is made narrower, as in
FIG. 12A, heat transfer from the bottom part 32 of the second heat
source 30 to the channel 70 becomes larger than that from the top
part 31 of the second heat source 30. Moreover, the high
denaturation temperature typical of the first heat source 20 is
more preferentially blocked in this embodiment as compared to the
embodiment with the top part of the second heat source 31 that is
made narrower, as in FIG. 12B.
[0312] In the examples shown in FIGS. 12A-B, the temperature
distribution of the channel 70 inside the second heat source 30 can
be controlled with the tapered chamber structure. Depending on the
temperature property of DNA polymerase used, the temperature
conditions inside the second heat source 30 may need to be adjusted
using such structure because the polymerization efficiency is
sensitive to the temperature conditions inside the second heat
source 30. For most widely used Taq DNA polymerase or its
derivatives, a first chamber wall 103 that is tapered from the top
to the bottom is more preferred since optimum temperature of Taq
DNA polymerase (around 70.degree. C.) is closer to the annealing
temperature compared to the denaturation temperature in typical
operation conditions.
[0313] C. One or Two Chambers, One Thermal Brake
[0314] Referring now to FIG. 4A, the apparatus 10 features the
first chamber 100 and the second chamber 110 disposed in the second
heat source 30 essentially symmetrically about the channel axis 80.
In this embodiment, the first chamber 100 is located on the bottom
part of the second heat source 30 and the second chamber 110 is
located on the upper part of the second heat source 30. The
apparatus 10 includes the first thermal brake 130 to help provide
more active control of the temperature distribution. In this
embodiment, the width of the first chamber 100 and the second
chamber 110 are about the same. However, the heights of the first
chamber 100 and the second chamber 110 can be varied between about
0.2 mm to about 80% or 90% of the length of the second heat source
30 along the channel axis 80, depending on the temperature property
of DNA polymerase used as discussed below. FIG. 4B provides an
expanded view of the first thermal brake 130 defined by the top end
131, bottom end 132, and wall 133 contacting the channel 70. In
this embodiment, the location and thickness of the first thermal
brake 130 along the channel axis 80 will be defined by the heights
of the first 100 and second 110 chambers along the channel axis 80.
The thickness of the thermal brake 130 along the channel axis 80 is
between about 0.1 mm to about 60% of the height of the second heat
source 30 along the channel axis 80, preferably between about 0.5
mm to about 40% of the height of the second heat source 30. The
first thermal brake 130 can be located nearly anywhere inside the
second heat source in between the first 100 and second 110
chambers, depending on temperature property of DNA polymerase used.
It is preferred to locate the first thermal brake 130 closer to the
bottom surface 32 of the second heat source 30 if optimum
temperature of DNA polymerase used is closer to the annealing
temperature of the second heat source 30 than the denaturation
temperature of the first heat source 20, or vice versa.
[0315] FIG. 13A is an example in which the first chamber 100 has a
smaller width than the second chamber 110, for instance, about 0.9
to about 0.3 times smaller, preferably about 0.8 to about 0.4 times
smaller. An opposite arrangement with the first chamber 100 having
a larger width than the second chamber 110 can also be used
depending on the temperature property of DNA polymerase used. An
expanded view of the first thermal brake 130 is shown in FIG.
13B.
[0316] In the embodiments shown in FIGS. 4A-B and 13A-B, the
apparatus features the first chamber and the second chamber that
are not tapered. In these embodiments, the first chamber is spaced
from the second chamber by a length (l) along the channel axis 80.
In one embodiment, the first chamber, the second chamber, and the
second heat source define a first thermal brake contacting the
channel between the first and second chambers with an area and a
thickness (or a volume) sufficient to reduce heat transfer from the
first heat source.
[0317] Referring to FIGS. 14A-B, the apparatus features the first
chamber 100 disposed symmetrically about the channel axis 80. The
first thermal brake 130 is positioned on the bottom of the second
heat source 30 between the first chamber 100 and the first
insulator 50.
[0318] The thickness of the first thermal brake 130 along the
channel axis 80 shown in FIGS. 14A-B is defined by distance from
the top end 131 to the bottom end 132 of the first thermal brake
130. Preferably that distance is between from about 0.1 mm to about
60% of the height of the second heat source 30 along the channel
axis 80, more preferably about 0.5 mm to about 40% of the height of
the second heat source 30.
[0319] In this embodiment, the apparatus features the first chamber
positioned on the bottom part of the second heat source and the
first chamber and the first insulator define the first thermal
brake. The first thermal brake contacts the channel between the
first chamber and the first insulator with an area and a thickness
(or a volume) sufficient to reduce heat transfer from the first
heat source. In this embodiment, the first thermal brake comprises
a top surface and a bottom surface in which the bottom surface of
the first thermal brake is located at about the same height as the
bottom surface of the second heat source. This embodiment is
particularly useful when using DNA polymerase that has optimum
temperature closer to the annealing temperature of the second heat
source than the denaturation temperature of the first heat source
(e.g., Taq DNA polymerase).
[0320] FIG. 14C is an example in which the chamber wall 103 of the
first chamber 100 is tapered from the top end 101 to the bottom end
102 of the first chamber 100. An opposite arrangement with the
chamber wall tapered from the bottom end 102 to the top end 101 of
the first chamber 100 can also be used depending on the temperature
property of DNA polymerase used. The first thermal brake 130 is
positioned on the bottom of the second heat source 30 between the
first chamber 100 and the first insulator 50. An expanded view of
the first thermal brake 130 is shown in FIG. 14D.
[0321] D. Asymmetric Receptor Hole
[0322] As mentioned, it is an object of the invention to provide an
apparatus with at least one temperature shaping element that has
horizontal asymmetry. By "horizontal asymmetry" is meant asymmetry
along a direction or plane perpendicular to the channel and/or
channel axis. It will be apparent that many of the apparatus
examples provided herein can be adapted to have a horizontal
asymmetry. In one embodiment, the receptor hole is placed
asymmetrically in the first heat source with respect to the channel
axis sufficient to generate a horizontally asymmetric temperature
distribution suitable for inducing a stable, directed convection
flow. Without wishing to be bound to theory, it is believed that
the region between the receptor hole and the bottom end of the
chamber is a location where a major driving force for thermal
convection flow can be generated. As will be readily apparent, this
region is where initial heating to the highest temperature (i.e.,
the denaturation temperature) and transition toward a lower
temperature (i.e., the polymerization temperature) take place, and
thus the largest driving force can originate from this region.
[0323] It is thus an object of the invention to provide an
apparatus with at least one horizontal asymmetry in which at least
one of the receptor holes (for instance, all of them) in the first
heat source has a width or diameter larger than the channel in the
first heat source. Preferably, the width disparity allows the
receptor hole to be off-centered from the channel axis. In this
example of the invention, the receptor hole asymmetry produces a
gap in which one side of the receptor hole is located closer to the
channel compared to the opposite side. It is believed that in this
embodiment, the apparatus will exhibit horizontally asymmetric
heating from the first heat source to the channel.
[0324] An example of such an invention apparatus is shown in FIG.
15. As shown, the receptor hole 73 is disposed asymmetrically with
respect to the channel axis 80 to form a receptor hole gap 74. That
is, the receptor hole 73 is slightly off-centered with respect to
the channel axis 80, for instance, by about 0.02 mm to about 0.5
mm. In this example, the receptor hole 73 has a width or diameter
perpendicular to the channel axis 80 that is larger than the width
or diameter of the channel 70. For example, the width or diameter
of the receptor hole 73 can be about 0.04 mm to about 1 mm larger
than the width or diameter of the channel 70.
[0325] Turning again to the embodiment shown in FIG. 15, one side
(the left side) of the channel 70 is in contact with the first heat
source 20 and the opposite side (the right side) is not in contact
with the first heat source 20 to form a receptor hole gap 74. While
the invention is compatible with several gap sizes, a typical
receptor hole gap can be as small as about 0.04 mm, particularly if
the other side is contacted to the channel. In other words, one
side is formed as a channel and the opposite side as a small space.
In this embodiment, it is believed that one side (the left side) is
heated preferentially over the opposite side (the right side),
providing a horizontally asymmetric heating directing the upward
flow to the preferentially heated side (the left side). A similar
effect can be obtained with a receptor hole having a gap from the
wall of the receptor hole that is smaller on one side than the
opposite side.
[0326] To enhance asymmetry, it is possible to make one side of the
receptor hole deeper than the other with respect to the first heat
source (and also closer to the chamber and the second heat source).
Referring now to the apparatus shown in FIGS. 16A-B, the receptor
hole 73 has a larger depth on one side of the hole (left side)
compared to the side opposite to the channel 70 (right side). In
this embodiment, both sides of the receptor hole 73 remain in
contact with the channel 70. As shown in FIG. 16A, the top portion
of the side wall of the receptor hole 73 is removed to form a
receptor hole gap 74 defined roughly by the channel 70 and the
first heat source 20. The bottom of the receptor hole gap 74 may be
perpendicular to the channel axis 80 (FIG. 16A) or it may be
disposed at an angle thereto (FIG. 16B). A side wall of the
receptor hole gap 74 may be parallel to the channel axis 80 (FIG.
16A) or it may be at an angle thereto (FIG. 16B). In both the
embodiments shown in FIGS. 16A-B, one side of the channel 70 has a
larger depth with respect to the first heat source 20 than the
other side with the receptor hole gap 74. Without wishing to be
bound to theory, it is believed that the channel side with the
larger depth in the embodiments shown in FIGS. 16A-B is heated
preferentially due to more heat transfer from the first heat
source, generating a larger buoyancy force on that side. It is
further believed that by adding such an asymmetric receptor hole 73
and receptor hole gap 74 to the apparatus, there is an increase of
the temperature gradient on one side of the channel 70 compared to
the opposite side (the temperature gradient is typically inversely
proportional to the distance). It is also believed that these
features create a larger driving force on one side (e.g., the left
side in FIGS. 16A and B) and support upward thermal convective flow
along that side. It will be appreciated that one or a combination
of different adaptations of the receptor hole 73 and receptor hole
gap 74 are possible to achieve this goal. However, for many
invention embodiments, it will be generally useful to make
difference in the receptor hole depth on two opposing sides in the
range of between from about 0.1 mm up to about 40 to 50% of the
receptor hole depth.
[0327] FIGS. 17A-B show further examples of suitable apparatus
embodiments in which the receptor hole 73 is disposed about the
channel asymmetrically. Portions of the receptor hole are deeper in
the first heat source and closer to the chamber or the second heat
source than other portions, thereby providing uneven thermal flow
toward the second heat source.
[0328] In the apparatus shown in FIG. 17A, the receptor hole 73 has
two surfaces coincident with the top 21 of the first heat source
20. Each surface faces the second heat source 30 and one of the
surfaces (the one on the right side in FIG. 17A) has a larger gap
on one side of the channel 70 compared to the surface opposite the
channel 70 (the one on the left side) with respect to the bottom
surface 32 of the second heat source 30. That is, one of the
surfaces is closer to the bottom 102 of the first chamber 100 or
the bottom surface 32 of the second heat source 30 than the other.
In this embodiment, both sides of the receptor hole 73 remain in
contact with the channel 70. The difference of the receptor hole
depth between the two surfaces is preferably in the range of
between from about 0.1 mm up to about 40 to 50% of the receptor
hole depth. The second heat source 30 features the first protrusion
33 that is disposed symmetrically about the channel axis 80. Also
in this embodiment, the first heat source 20 includes the first
protrusion 23 disposed asymmetrically about the channel axis
80.
[0329] Turning to FIG. 17B, the receptor hole 73 has a single
inclined surface coincident with the top 21 of the first heat
source 20. The incline angle is between about 2.degree. to about
45.degree. with respect to an axis perpendicular to the channel
axis 80. In this embodiment, the apex of the inclined surface is
relatively close to the bottom 102 of the first chamber 100. The
second heat source 30 features the first protrusion 33 that is
disposed symmetrically about the channel axis 80. Also in this
embodiment, the first heat source 20 includes the first protrusion
23 disposed asymmetrically about the channel axis 80.
[0330] E. One Asymmetric Chamber, Asymmetric or Symmetric Receptor
Hole
[0331] In the embodiment shown in FIG. 18A-B, the first chamber 100
is disposed asymmetrically about the channel axis 80 sufficient to
cause horizontally uneven heat transfer from the second heat source
20 to the channel 70. The receptor hole 73 may also be disposed
asymmetrically about the channel 70 as in FIGS. 18A-B. In the
embodiment shown in FIG. 18A, the first chamber 100 is positioned
within the second heat source 30 and has a greater height on one
side of the chamber than the other side opposite the channel axis
80. That is, the length between one surface of the top end of the
first chamber 101 and one surface of the bottom end of the first
chamber 102 is greater (left side of FIG. 18A) along the channel
axis 80 than the length between another surface of the top end of
the first chamber 101 and another surface of the bottom end of the
first chamber 102 (right side of FIG. 18A). The difference of the
chamber height between the two opposing sides is preferably in the
range of between from about 0.1 mm up to about 5 mm. There is gap
between the bottom 101 of the first chamber 100 (or the bottom
surface of the second heat source) and the top end of the receptor
hole 73 that is smaller on the left side of the channel 70 than the
other side.
[0332] Turning to FIG. 18B, the bottom end 102 of the first chamber
100 is inclined with respect to an axis perpendicular the channel
axis 80 by from about 2.degree. to about 45.degree.. In the
example, the apex of the incline is further closer to the receptor
hole 73. The top of the receptor hole 73 coincident with the top
surface 21 of the first heat source 20 is inclined with respect to
the channel axis 80. In this embodiment, the apex of the receptor
hole incline is closer to the bottom end of the first chamber 102.
That is, there is gap between the bottom of the first chamber 100
(or the bottom surface of the second heat source) and the top end
of the receptor hole 73 that is smaller on the left side of the
channel 70 than the other side.
[0333] The configurations shown in FIGS. 18A-B provide preferential
heating on one side of the channel 70 (i.e., the left side) in the
receptor hole 73, and thus initial upward convective flow can start
preferentially on that side. However, the second heat source 30
provides preferential cooling on the same side due to the longer
chamber length on that side. Therefore, the upward flow can change
its path to the other side depending on the extent of the first
chamber asymmetry.
[0334] Turning to FIGS. 18C-D, the length between the top end 101
and the bottom end 102 is greater on one side of the first chamber
100 (the right side) than the other side with respect to the
channel axis 80. Here, preferential cooling from the second heat
source will be on the right side of the chamber shown in FIGS.
18C-D. Further asymmetry is provided by the larger depth of the
receptor hole 73 on one side of the channel 70 (i.e., the left side
of FIGS. 18C-D) than the other side. In the receptor hole 73,
preferential heating will be on the left side of the channel 70. In
this embodiment, a gap between the bottom 102 of the chamber 100
and the top of the receptor hole 73 is essentially constant around
the channel 70.
[0335] The configurations shown in FIGS. 18C-D support preferential
heating on one side of the channel 70 (i.e., the left side) in the
receptor hole 73 and preferential cooling on the opposite side in
the first chamber 100, and thus upward convective flow will stay
preferentially on the left side.
[0336] In the embodiments shown in FIGS. 18A-D, asymmetry
introduced by the chamber configurations is sufficient to cause
horizontally uneven heat transfer from the second heat source to
the channel. Also in these embodiments, the protrusions 23, 33 are
disposed asymmetrically about the channel axis 80.
[0337] Other apparatus embodiments with at least one structural
asymmetry are within the scope of the present invention.
[0338] For example, and as shown in FIGS. 19A-B, the bottom end of
the first chamber 102, is asymmetrically disposed with respect to
the channel axis 80. The length between the top end 101 and the
bottom end 102 is greater on one side of the first chamber 100 (the
left side of the FIGS. 19A-B) than the other side with respect to
the channel axis 80. A gap between the bottom of the first chamber
102 and the top of the receptor hole 73 is smaller on one side of
the channel 70 (the left side of FIGS. 19A-B) than the other side.
In these embodiments, the first protrusion 23 of the first heat
source 20 is disposed symmetrically about the channel axis 80. Also
in these embodiments, there is preferential heating on the right
side of the receptor hole 73 (with respect to the channel axis 80)
due to the larger gap on that side (since cooling by the second
heat source is less significant on that side due to the larger gap)
and thus a larger driving force is generated on the right side of
the channel 70 and more pronounced upward flow on that side. In
addition, the second heat source 30 features a first protrusion 33
disposed asymmetrically about the channel axis 80.
[0339] F. One Asymmetric Chamber with or without Thermal Brake
[0340] Referring to FIG. 20A, the first chamber 100 is off-centered
with respect to the channel axis 80. In this embodiment, the
receptor hole 73 is disposed symmetrically about the channel axis
80 and is of constant depth. The first chamber 100 is off-centered
from the channel 70 so that the chamber gap 105 is smaller on one
side compared to the opposite side. As shown in FIG. 20B, the
chamber 100 can be further off-centered from the channel 70 so that
one side or wall of the channel 70 makes contact with the chamber
wall. In this embodiment, the channel-forming side (e.g., the left
side in FIG. 29B) functions as a first thermal brake 130 having its
top 131 and bottom 132 ends coincide with the top 101 and bottom
102 end of the first chamber 100. In such an embodiment, heat
transfer between the second heat source 30 and the channel 70 is
larger on the side where the chamber gap 105 is smaller or does not
exist (i.e., the left side in FIGS. 20A and B), thus producing a
horizontally asymmetric temperature distribution. FIG. 20C provides
an expanded view of the first thermal brake 130. An acceptable
difference between the chamber gaps on two opposite sides is
preferably in the range between from about 0.2 mm to about 4 to 6
mm, and hence the chamber axis is off-centered from the channel
axis by at least about 0.1 mm up to about 2 to 3 mm.
[0341] It will be appreciated that all or part of a chamber can be
made asymmetric with respect to the channel axis 80, for example,
all or part of the chamber may be off-centered. For most invention
applications, it will be useful to off-center an entire
chamber.
[0342] G. Asymmetric Chambers
[0343] As discussed, it is an object of the present invention to
provide an apparatus within one, two or three chambers in the
second heat source, for example. In one embodiment, at least one of
the chambers has a horizontal asymmetry. The asymmetry helps create
a horizontally asymmetric driving force within the apparatus. For
example, and in the embodiment shown in FIG. 21, the first chamber
100 and the second chamber 110 are each off-centered from the
channel axis 80 along opposite directions. In particular, the top
end of the first chamber 101 is positioned at essentially at the
same height as the bottom end of the second chamber 112. The first
and second chambers may have different width or diameter.
Difference of the chamber gap 105, 115 on two opposite sides may be
at least about 0.2 mm up to about 4 to 6 mm.
[0344] In addition to the off-centered chamber structures
exemplified in FIG. 21, one or more of the chambers may be made
horizontally asymmetric by including structures that are tilted
(skewed) with respect to the channel axis 80. For instance, and as
shown in FIG. 22, the first chamber 100 may be tilted with respect
to the channel axis 80. In this embodiment, the first wall of the
first chamber 103 is tilted with respect to the channel axis 80
(e.g., at an angle less than about 30.degree. with respect to the
channel axis 80). Tilt angle as defined by an angle between the
center axis of the chamber (or the chamber wall 103) and the
channel axis may be between from about 2.degree. to about
30.degree., more preferably between from about 5.degree. to about
20.degree..
[0345] In the apparatus embodiments shown in FIGS. 21 and 22,
upward convective flow from the bottom of the channel 70 is favored
along the right side of the channel 70 as a result of preferential
heating from the receptor hole 73 on that side (due to less
significant cooling by the second heat source as a result of the
larger chamber gap on that side).
[0346] H. One Chamber in Second Heat Source, Tilted
[0347] As mentioned, it is an object of the invention to provide an
apparatus in which various temperature shaping elements such as one
or more of the channel, receptor hole, protrusion (if present), gap
such as a chamber, insulators or insulating gaps, and thermal brake
are each disposed symmetrically about the channel axis. In use, the
apparatus will often be placed on a flat, horizontal surface so
that the channel axis will be substantially aligned with the
direction of gravity. In such an orientation, it is believed that a
buoyancy force is generated by the temperature gradient inside the
channel and that the buoyancy force also becomes aligned parallel
to the channel axis. It is also believed that the buoyancy force
will have its direction opposite to the direction of gravity with a
magnitude proportional to the temperature gradient (along the
vertical direction). Since the channel and the one or more chambers
are symmetrically disposed about the channel axis in this
embodiment, it is believed that the temperature distribution (i.e.,
distribution of the temperature gradient) generated inside the
channel should also be symmetric with respect to the channel axis.
Therefore, distribution of the buoyancy force should also be
symmetric with respect to the channel axis with its direction
parallel to the channel axis.
[0348] It is possible to introduce a horizontal asymmetry into the
apparatus by moving the channel axis away from the direction of
gravity. In these embodiments, it is possible to further enhance
the efficiency and speed of convection-based PCR within the
apparatus. Thus it is an object of the invention to provide an
apparatus featuring one or more horizontal asymmetries.
[0349] Examples of an invention apparatus with positional
horizontal asymmetry are provided by FIGS. 11A-B.
[0350] In FIG. 11A, the channel axis 80 is offset with respect to
the direction of gravity to give the apparatus a positional
horizontal asymmetry. In particular, the channel and chamber are
formed symmetrically with respect to the channel axis. However the
whole apparatus is rotated (or tilted) by an angle .theta..sub.g
with respect to the direction of gravity. In this tilted structure,
the channel axis 80 is no longer parallel to the direction of
gravity, and thus the buoyancy force generated by the temperature
gradient on the bottom of the channel becomes tilted with respect
to the channel axis 80 since it is supposed to have a direction
opposite to the direction of gravity. Without wishing to be bound
to theory, the direction of the buoyancy force makes an angle
.theta..sub.g with the channel axis 80 even if the channel/chamber
structure is symmetric with respect to the channel axis 80. In this
structural arrangement, the upward convection flow will take a
route on one side of the channel or the reaction vessel (the left
side in the case of FIG. 11A) and the downward flow will take a
route on the opposite side (i.e., the right side in the case of
FIG. 11A). Hence, the route or pattern of the convection flow is
believed to become substantially locked to one determined by such
structural arrangement, therefore the convective flow becomes more
stable and not sensitive to small perturbations from environment or
small structural defects, leading to more stable convection flow
and enhanced PCR amplification. It has been found that introduction
of the gravity tilting angle helps enhancing the speed of the
thermal convection, thereby supporting faster and more robust
convection PCR amplification. The tilt angle .theta..sub.g can be
varied between from about 2.degree. to about 60.degree., preferably
between about 5.degree. to about 30.degree.. This tilted structure
can be used in combination with all the symmetric or asymmetric
channel/chamber structures provided in the present invention.
[0351] The tilt angle .theta..sub.g shown in FIG. 11A can be
introduced by one or a combination of different element. In one
embodiment, the tilt is introduced manually. However it will often
be more convenient to introduce the tilt angle .theta..sub.g by
placing the apparatus 10 on an incline, for instance, by placing
apparatus 10 on a wedge or similar shaped base.
[0352] However for some invention embodiments, it will not be
useful to tilt the apparatus 10. FIG. 11B shows another approach
for introducing the horizontal asymmetry. As shown, one or more of
the channel and chambers is tilted with respect to the direction of
gravity. That is, the channel axis 80 (and the chamber axis) are
offset (by .theta..sub.g) with respect to an axis perpendicular to
the horizontal surface of the heat sources. In this invention
embodiment, the channel axis 80 makes an angle .theta..sub.g with
respect to the direction of gravity when the apparatus is placed on
a flat, horizontal surface to have its bottom opposite from and
parallel to that surface (as would be typical). According to this
embodiment, and without wishing to be bound to theory, the buoyancy
force generated by the temperature gradient on the bottom of the
channel (that is supposed to have a direction opposite to the
direction of gravity) will make an angle .theta..sub.g with respect
to the channel axis as in the case of the embodiments described
above. Such a structural arrangement will make the convection flow
going upward on one side (i.e., the left side in the case of FIG.
11B) and going downward on the opposite side (i.e., the right side
in the case of FIG. 11B). The tilt angle .theta..sub.g can be
varied preferably between from about 2.degree. to about 60.degree.,
more preferably between about 5.degree. to about 30.degree.. This
tilted structure can also be used in combination with all the
structural features of the channel and the chamber provided in the
present invention.
[0353] Nearly any of the apparatus embodiment disclosed herein can
be tilted by placing it on a structure capable of offsetting the
channel axis 80 between from about 2.degree. to about 60.degree.
with respect to the direction of gravity. As mentioned, an example
of an acceptable structure is a surface capable of producing an
incline such as a wedge or related shape.
[0354] L. Two Chambers and Thermal Brake(s) with Structural
Asymmetry
[0355] It is an object of the invention to provide an apparatus
with one or more thermal brakes, e.g., one, two or three thermal
brakes in which one or more of them have horizontal asymmetry.
Referring to the apparatus shown in FIGS. 23A-B, the first thermal
brake 130 has horizontal asymmetry. In this embodiment, the through
hole formed in the first thermal brake 130 (that typically is made
to fit with the channel) is larger than the channel 70 and
off-centered from the channel axis 80 to provide a smaller (or no)
gap on one side and a larger gap on the opposite side. Temperature
distribution is found to be more sensitive to the asymmetry in the
thermal brake compared to the asymmetry in the chamber (i.e.,
asymmetry in the first chamber wall 103). Preferably, the through
hole in the thermal brake may be made at least about 0.1 mm up to
about 2 mm larger, and off-centered from the channel axis by at
least about 0.05 mm up to about 1 mm.
[0356] In embodiments in which the structural asymmetry resides in
the first thermal brake 130 or the second thermal brake 140 (or
both the first 130 and second 140 thermal brakes), the apparatus
can include at least one chamber that is disposed symmetrically or
asymmetrically about the channel axis 80. In the embodiment shown
in FIG. 23A, the first chamber 100 and the second chamber 110 are
positioned within the second heat source 30 and disposed
symmetrically about the channel axis 80. In this embodiment, the
first chamber 100 is spaced from the second chamber 110 by a length
l along the channel axis 80. A portion of the second heat source 30
contacts the channel 70 to form the first thermal brake 130
sufficient to reduce heat transfer from the first heat source 20.
The first thermal brake 130 is disposed asymmetrically about the
channel 70. The first thermal brake 130 contacts one side of the
channel 70 between the first 100 and second 110 chambers, the other
side of the channel 70 being spaced from the second heat source 30.
FIG. 23B shows an expanded view of the first thermal brake 130
showing wall 133 contacting the channel 70 on the left side. When
the structural asymmetry is associated with one or more of the
thermal brakes, the upward and downward convective flow can be
favored on one side of the channel or the opposite side with
respect to the channel axis depending on the position and thickness
of the thermal brakes along the channel axis.
[0357] It will sometimes be useful to have an invention apparatus
with one, two, or three chambers disposed in the second heat source
either symmetrically or asymmetrically about the channel axis 80.
In one embodiment, the apparatus has a first, second, and third
chamber in which one or two of the chambers is disposed
asymmetrically about the channel axis 80 and the other chamber is
disposed symmetrically about the same axis. In an embodiment in
which the apparatus includes a first chamber and second chamber
that are each disposed asymmetrically about the channel axis 80,
those chambers can reside completely or partially within the second
heat source.
[0358] Particular examples of this invention embodiment are shown
in FIGS. 24A-D.
[0359] In FIG. 24A, the first thermal brake 130 contacts part of
the height of the channel 70 within the second heat source 30. The
first chamber 100 and the second chamber 110 are each positioned in
the second heat source 30 and the first chamber 100 is spaced from
the second chamber 110 by a length (l) along the channel axis 80.
In this embodiment, the thermal brake 130 contacts the whole
circumference of the channel 70 on the length (l) between the first
100 and second 110 chambers. The first chamber 100 and the second
chamber 110 are each off-centered from the channel axis 80 in the
same horizontal direction. FIG. 24B provides an expanded view of
the first thermal brake 130 in which wall 133 contacts the channel
70.
[0360] Turning to FIG. 24C, the first chamber 100 and the second
chamber 110 are each off-centered from the channel axis in the same
horizontal direction. The first 100 and second 110 chambers can
have the same or different width or diameter. In this embodiment,
the first thermal brake 130 contacts one side of the channel 70
(i.e., the left side) within the first chamber 100 on a length from
the bottom end 132 to the top end 131 of the first thermal brake
130 that is the same as the length of the first chamber 100 along
the channel axis 80 in the embodiment shown in FIG. 24C. FIG. 24D
provides an expanded view of the first thermal brake 130 showing
wall 133 contacting the channel 70.
[0361] In each of the embodiments shown in FIGS. 24A-D, the
receptor hole 73 is disposed symmetrically about the channel
70.
[0362] FIG. 25A shows an invention embodiment in which the first
chamber 100 and the second chamber 110 are each off-centered in
opposite directions with respect to the channel axis 80 by about
0.1 mm up to about 2 to 3 mm. The first thermal brake 130 is
symmetrically disposed with respect to the channel axis 80. In this
embodiment, a portion of the second heat source 30 contacts the
channel 70 to form a first thermal brake 130 sufficient to reduce
heat transfer from the first heat source 20. In this example of the
invention, the first thermal brake 130 contacts the whole
circumference of the channel 70 on a length (l) between the first
100 and second 110 chambers. In other embodiments, the first
thermal brake 130 can contact the channel 70 on one side, the other
side being spaced from the second heat source 30. FIG. 25B provides
an expanded view of the first thermal brake 130 showing wall 133
contacting the channel 70.
[0363] Referring to the embodiment shown in FIG. 26A, the first
chamber 100 and second chamber 110 are each off-centered with
respect to the channel axis 80 in the same horizontal direction
(e.g., by about 0.1 mm up to about 2 to 3 mm). In this embodiment,
the first thermal brake 130 is asymmetrically disposed with respect
to the channel axis 80. The first thermal brake 130 and the chamber
wall 103 are off-centered to the same direction. In this
embodiment, the first thermal brake 130 contacts the channel 70 on
one side (i.e., the left side), the other side being spaced from
the second heat source 30. FIG. 26B shows an expanded view of the
first thermal brake 130.
[0364] In FIG. 26C, the first chamber 100 and the second chamber
110 are each off-centered with respect to the channel axis 80 in
the same horizontal direction and the first thermal brake 130 is
off-centered to the opposite direction. In this embodiment, the
first thermal brake 130 contacts the channel 70 on one side (i.e.,
the right side), the other side being spaced from the second heat
source 30. FIG. 26D shows an expanded view of the first thermal
brake 130.
[0365] In another invention embodiment, the apparatus has two
chambers in the second heat source 30 in which each chamber is
off-set from the other in different horizontal directions. FIG. 27A
shows an example. Here, the first chamber 100 and second chamber
110 within the second heat source 30 are each off-set with respect
to the channel axis 80 in opposite horizontal directions (e.g., by
about 0.5 mm to about 2 to 2.5 mm). The wall of the first chamber
103 is disposed lower along the channel axis 80 than the wall of
the second chamber 113. The wall of the first thermal brake 133
contacts one side of the channel 70 (i.e., the left side) on the
lower part of the channel 70 within the first chamber 100, and the
wall of the second thermal brake 143 contacts the other side of the
channel (i.e., the right side) on the upper part of the channel 70
within the second chamber 110. The top end of the first thermal
brake 131 is positioned essentially at the same height as the
bottom end of the second thermal brake 142. This arrangement is
generally sufficient to cause horizontally uneven heat transfer
between the second heat source 30 and the channel 70. FIG. 27B
shows an expanded view of the first thermal brake 130 and the
second thermal brake 140.
[0366] FIG. 27C shows an invention embodiment in which the top end
of the first thermal brake 131 is positioned higher than the bottom
end of the second thermal brake 142. The wall of the first thermal
brake 133 and the wall of the second thermal brake 143 each contact
the channel 70 on one side. FIG. 27D shows an expanded view of the
first thermal brake 130 and the second thermal brake 140.
[0367] FIG. 27E shows an embodiment in which the top end of the
first thermal brake 131 is positioned lower than the bottom end of
the second thermal brake 142. The wall of the first thermal brake
133 and the wall of the second thermal brake 143 each contact the
channel 70 on one side. FIG. 27F shows an expanded view of the
first thermal brake 130 and the second thermal brake 140.
[0368] The invention provides other embodiments in which an
asymmetry is introduced into the apparatus by tilting (skewing) one
or more of the thermal brakes or the chamber with respect to the
channel axis. Referring now to FIG. 28A, the top end of the first
chamber 101 and the bottom end of the second chamber 112 are each
inclined between about 2.degree. to about 45.degree. with respect
to an axis perpendicular to the channel axis 80. In this
embodiment, the distance between the top end of the first heat
source 21 and the bottom end of the first thermal brake 132 is
smaller on one side (i.e., the left side) with respect to the
channel axis 80, resulting in a temperature gradient that is biased
to be larger on that side of the first chamber 100. The thermal
brake 130 contacts the whole circumference of the channel 70
between the first chamber 100 and the second chamber 110 and at a
higher location on one side than the other side. FIG. 28B shows an
expanded view of the first chamber 100, first thermal brake 130 and
the second chamber 110 in which wall 133 contacts the channel
70.
[0369] In some invention embodiments, it will be useful to tilt at
least one of the chambers with respect to the channel axis (e.g.,
one, two, or three of the chambers). Indeed, different combinations
of the tilted or skewed structures may be adopted to achieve the
intended horizontally asymmetric temperature distribution. A few
examples are shown in FIGS. 29A-D.
[0370] In particular, FIG. 29A shows a case in which the first
chamber 100 and the second chamber 110 are each tilted or skewed
with respect to the channel axis 80 between about 2.degree. to
about 30.degree.. In this embodiment, the first thermal brake 130
is not tilted. FIG. 29B shows an expanded view of the first chamber
100, the first thermal brake 130 and the second chamber 110 in
which wall 133 contacts the channel 70.
[0371] FIG. 29C shows an example in which both of the first chamber
100, the second chamber 110, and the first thermal brake 130 are
each tilted with respect to the channel axis 80. Each of the first
chamber 100 and the second chamber 110 can be tilted or skewed with
respect to the channel axis 80 by between about 2.degree. to about
30.degree.. The top end 131 and bottom end 132 of the first thermal
brake 130 can be each inclined or tilted by between about 2.degree.
to about 45.degree. with respect to an axis perpendicular to the
channel axis 80. In this embodiment, the first thermal brake 130
contacts the whole circumference of the channel between the first
chamber and the second chamber and at a higher location on one side
than the other side.
[0372] In the embodiments shown in FIGS. 25A-B, 26A-D, 27A-F,
28A-B, and 29A-D, the receptor hole 73 is disposed symmetrically
about the channel axis 80.
[0373] Manufacture, Use and Temperature Shaping Element
Selection
[0374] A. Heat Sources
[0375] For most invention embodiments, one or more of the heat
sources can be made with materials having a relatively low thermal
conductivity as compared to materials used for other thermal
cycling type apparatuses. Rapid temperature changing process can be
usually avoided in the present invention. Therefore, a high
temperature uniformity across each of the heat sources (e.g., with
a temperature variation smaller than about 0.1.degree. C.) can be
readily achieved using a material having a relatively low thermal
conductivity. The heat sources can be made of any solid material
that has a thermal conductivity sufficiently larger than that of
the sample or the reaction vessel, for instance, preferably at
least about 10 times larger, more preferably at least about 100
times larger. The sample to be heated is mostly water that has a
thermal conductivity of 0.58 Wm.sup.-1K.sup.-1 at room temperature,
and the reaction vessel is typically made of a plastic that has a
thermal conductivity typically about a few tenths of
Wm.sup.-1K.sup.-1. Therefore, the thermal conductivity of a
suitable material is at least about 5 Wm.sup.-1K.sup.-1 or larger,
more preferably at least about 50 Wm.sup.-1K.sup.-1 or larger. If
the reaction vessel is made of a glass or ceramic that has a
thermal conductivity larger than that of a plastic, it is preferred
to use a material having somewhat larger thermal conductivity, for
instance one having a thermal conductivity larger than about 80 or
about 100 Wm.sup.-1K.sup.-1. Most metals and metal alloys as well
as some high thermal conductivity ceramics fulfill such
requirement. Although materials having a higher thermal
conductivity will generally provide better temperature uniformity
across each of the heat sources, aluminum alloys and copper alloys
are typically useful materials since they are relatively cheap and
easy to fabricate while possessing high thermal conductivity.
[0376] The following specifications will be generally useful for
making and using apparatus embodiments described herein. The width
and length dimensions of the first and second heat sources along an
axis perpendicular to the channel axis can be selected as any
values depending on intended use, for instance, depending on
spacing between adjacent channel/chamber structures. The spacing
between the adjacent channel/chamber structures can be at least
about 2 to 3 mm, preferably between about 4 mm to about 15 mm. It
will be generally preferred to use the industry standards, i.e.,
4.5 mm or 9 mm spacing. In typical embodiments, the channel/chamber
structures are arranged in equally spaced rows and/or columns. In
such embodiments, it is preferred to make the width or length
(along an axis perpendicular to the channel axis) of each of the
heat sources that is at least about the value corresponding to the
spacing times the number of rows or columns up to about one to
about three spacing larger than this value. In other embodiments,
the channel/chamber structures may be arranged in a circular
pattern and preferably equally spaced. The spacing in such
embodiments is also at least about 2 to 3 mm, preferably about 4 mm
to about 15 mm with the industry standards of 4.5 mm or 9 mm
spacing more preferred. In these embodiments, it is preferred to
have the shape of the heat sources as a donut-like shape typically
having a hole in the center. The channel/chamber structures may be
positioned on one, two, three, up to about ten concentric circles.
Diameter of each concentric circle can be determined by a geometric
requirement for intended use, e.g., depending on number of the
channel/chamber structures, spacing between adjacent
channel/chamber structures in that circle, etc. Outer diameter of
the heat sources is preferably at least about one spacing larger
than diameter of the largest concentric circle, and inner diameter
of the heat sources is preferably at least about one spacing
smaller than diameter of the smallest concentric circle.
[0377] Length or thickness of the first and second heat sources
along the channel axis has been already discussed. In the
embodiments comprising at least one chamber in the second heat
source, the thickness of the first heat source is larger than about
1 mm along the channel axis, preferably from about 2 mm to about 10
mm. Thickness of the second heat source along the channel axis is
between about 2 mm to about 25 mm, preferably between 3 mm to about
15 mm.
[0378] The channel dimensions can be defined by a few parameters as
denoted in FIGS. 7A-D and 8A-J. The height (h) of the channel along
the channel axis is at least about 5 mm to about 25 mm, preferably
8 mm to about 16 mm for a sample volume of about 20 microliters.
The taper angle (.theta.) is between from about 0.degree. to about
15.degree., preferably from about 2.degree. to about 10.degree..
The width (w1) or diameter of the channel (or its average) along an
axis perpendicular to the channel axis is at least about 1 mm to
about 5 mm. The vertical aspect ratio as defined by the ratio of
the height (h) to the width (w1) is between about 4 to about 15,
preferably from about 5 to about 10. The horizontal aspect ratio as
defined by the ratio of the first width (w1) to the second width
(w2) along first and second directions, respectively, that are
mutually perpendicular to each other and aligned perpendicular to
the channel axis, is typically between about 1 to about 4.
[0379] The receptor hole has a width or diameter that is in the
same range as the channel, i.e., at least about 1 mm to about 5 mm.
When the channel is tapered, the width or diameter of the receptor
hole is smaller or larger than that of the channel depending on the
tapering direction. Depth of the receptor hole is typically at
least about 0.5 mm up to about 8 mm, preferably between about 1 mm
to about 5 mm.
[0380] The chamber typically has a width or diameter along an axis
perpendicular to the channel axis that is at least about 1 mm to
about 10 or 12 mm, preferably between about 2 mm to about 8 mm.
Presence of the chamber structure provide the chamber gap between
the channel and the chamber wall that is typically between about
0.1 mm to about 6 mm, more preferably about 0.2 mm to about 4 mm.
Length or height of the chamber along the channel axis can vary
depending on different embodiments. For instance, if the apparatus
comprises one chamber in the second heat source, that chamber can
have a height along the channel axis between about 1 mm to about 25
mm, preferably between about 2 mm to about 15 mm. In the
embodiments having two or more chambers in the second heat source,
the height of each chamber is between about 0.2 mm to about 80% or
90% of the thickness of the second heat source along the channel
axis.
[0381] Dimensions of the thermal brake and the insulators (or
insulating gaps) are also very important. Please refer to the
general specifications as already provided above.
[0382] Although not generally required for optimal use of the
invention, it is within the scope of the present invention to
provide an apparatus with protrusions 24, 34, or both. See FIG. 6A,
for example.
[0383] It will be appreciated that there usually exists certain
tolerance in machining or fabricating mechanical structures.
Therefore, in actual practice, the physically contacting holes
(e.g., the through hole in the second heat source or the receptor
hole in the first heat source in particular embodiments) must be
designed to have a positive tolerance with respect to the size of
the reaction vessel. Otherwise, the through hole or the channel
could be made smaller or equal to the size of the reaction vessel,
not allowing proper installation of the reaction vessel to the
channel. Practically reliable tolerance for the physically
contacting hole is about +0.05 mm in standard fabrication process.
Therefore, if two objects are said to be "in physical contact", it
should be interpreted as having a gap between the two contacting
objects that is smaller than or equal to about 0.05 mm. If two
objects are said to be "not in physical contact", or "spaced", it
should be interpreted as having a gap between the two objects that
is larger than about 0.05 or 0.1 mm.
[0384] B. Use
[0385] Nearly any thermal convection PCR apparatus described herein
can be used to perform one or a combination of different PCR
amplification techniques. One suitable method includes at least one
of and preferably all of the following steps: [0386] (a)
maintaining a first heat source comprising a receptor hole at a
temperature range suitable for denaturing a double-stranded nucleic
acid molecule and forming a single-stranded template, [0387] (b)
maintaining a second heat source at a temperature range suitable
for annealing at least one oligonucleotide primer to the
single-stranded template; and [0388] (c) producing thermal
convection between the receptor hole and second heat source under
conditions sufficient to produce the primer extension product.
[0389] In one embodiment, the method further includes the step of
providing a reaction vessel comprising the double-stranded nucleic
acid and the oligonucleotide primer(s) in aqueous buffer solution.
Typically, the reaction vessel further includes one or more DNA
polymerases. If desired, the enzyme may be immobilized. In a more
particular embodiment of the reaction method, the method includes a
step of contacting (either directly or indirectly) the reaction
vessel to the receptor hole, the through hole, and at least one
temperature shaping element (typically at least one chamber)
disposed within at least one of the second or first heat sources.
In this embodiment, the contacting is sufficient to support the
thermal convection within the reaction vessel. Preferably, the
method further includes a step of contacting the reaction vessel to
a first insulator between the first and second heat sources. In one
embodiment, the first and second heat sources have a thermal
conductivity at least about tenfold, preferably about one hundred
fold greater than the reaction vessel or aqueous solution therein.
The first insulator may have a thermal conductivity at least about
five fold smaller than the reaction vessel or aqueous solution
therein in which the thermal conductivity of the first insulator is
sufficient to reduce heat transfer between the first and second
heat sources.
[0390] In the step (c) of the foregoing method, the thermal
convection fluid flow is produced essentially symmetrically or
asymmetrically about the channel axis within the reaction vessel.
Preferably, the steps (a)-(c) of the method described above consume
less than about 1 W, preferably less than about 0.5 W of power per
reaction vessel to produce the primer extension product. If
desired, the power for performing the method is supplied by a
battery. In typical embodiments, the PCR extension product is
produced in about 15 to about 30 minutes or shorter and the
reaction vessel can have a volume of less than about 50 or 100
microliters, for example, less than or equal to about 20
microliters.
[0391] In embodiments in which the method is used with a thermal
convection PCR centrifuge of the invention, the method further
includes the step of applying or impressing a centrifugal force to
the reaction vessel conducive to performing the PCR.
[0392] In a more specific embodiment of the method for performing
PCR by thermal convection, the method includes the steps of adding
an oligonucleotide primer, nucleic acid template, and buffer to a
reaction vessel received by any of the apparatuses disclosed herein
under conditions sufficient to produce a primer extension product.
In one embodiment, the method further comprises a step of adding a
DNA polymerase to the reaction vessel.
[0393] In another embodiment of a method for performing PCR by
thermal convection, the method comprising the steps of adding an
oligonucleotide primer, nucleic acid template, and buffer to a
reaction vessel received by any PCR centrifuge disclosed herein and
applying a centrifugal force to the reaction vessel under
conditions sufficient to produce a primer extension product. In one
embodiment, the method includes the step of adding a DNA polymerase
to the reaction vessel.
[0394] Practice of the invention is compatible with one or a
combination of PCR techniques including quantitative PCR (qPCR),
multiplex PCR, ligation-mediated PCR, hot-start PCR,
allele-specific PCR among other variations of the amplification
technique. The following particular use of the invention is with
reference to the embodiment shown in FIGS. 1 and 2A. As will be
appreciated however, the methodology is generally applicable to
other embodiments referred to herein.
[0395] Referring to FIGS. 1 and 2A, the first heat source 20
generates a temperature distribution suitable for the denaturation
process on the bottom or lower portion of the channel (sometimes
referred herein to as a denaturation region). The first heat source
20 is typically maintained at a temperature useful to melt the
nucleic acid template of interest (e.g., about 1 fg to about 100 ng
of a DNA-based template). In this embodiment, the first heat source
20 should be maintained at between about 92.degree. C. to about
106.degree. C., preferably between about 94.degree. C. to about
104.degree. C., and more preferably between about 96.degree. C. to
about 102.degree. C. As will be appreciated, other temperature
profiles may be better suited for optimal practice of the invention
depending on recognized parameters such as the nucleic acid of
interest, the sensitivity desired, and the speed of which the PCR
process should be conducted.
[0396] The second heat source 30 generates a temperature
distribution suitable for the annealing process on the top or upper
portion of the channel (sometimes referred herein to as an
annealing region). The second heat source is typically maintained
at a temperature between about 45.degree. C. to about 65.degree.
C., depending, for instance, on the melting temperatures of the
oligonucleotide primers used and other parameters known to those
with experience in PCR reactions.
[0397] A temperature distribution suitable for the polymerization
process is generated in the intermediate region (i.e., transition
region) of the channel 70 (sometimes referred herein to as a
polymerization region) in between the denaturation region on the
bottom of the channel and the annealing region on the top or upper
part of the channel. For some instances (in which the temperature
of the second heat source is maintained at a temperature equal to
or higher than about 60.degree. C.), the annealing region on the
top part of the channel can also function as part of the
polymerization region. For many invention applications, the
polymerization region is typically maintained at a temperature
between about 60.degree. C. to about 80.degree. C., more preferably
between about 65.degree. C. to about 75.degree. C., in cases in
which Taq DNA polymerase or a relatively heat stable derivative
thereof is used. If a DNA polymerase that has a different
temperature profile of its activity is used, the temperature range
of the polymerization region can be changed (by changing the
annealing temperature of the second heat source or the structure of
the temperature shaping elements) to match with the temperature
profile of the polymerase used. See U.S. Pat. No. 7,238,505 and
references disclosed therein regarding use of heat sensitive and
heat stable polymerases in the PCR process.
[0398] See the Examples section for information about use of
additional apparatus embodiments.
[0399] C. Selection of Temperature Shaping Elements
[0400] The following section is intended to provide further
guidance on the selection and use of temperature shaping elements.
It is not intended to limit the invention to a particular apparatus
design or use.
[0401] Choice of one or a combination of temperature shaping
elements for use with an invention apparatus will be guided by the
particular PCR application of interest. For instance, properties of
the target template are important for selecting temperature shaping
element(s) that is/are best suited for a particular PCR
application. For instance, the target sequence may be relatively
short or long; and/or the target sequence may have a relatively
simple structure (such as in plasmid or bacterial DNA, viral DNA,
phage DNA, or cDNA) or a complex structure (such as in genomic or
chromosomal DNA). In general, target sequences having longer
sequences and/or complex structures are more difficult to amplify
and typically require a longer polymerization time. Additionally,
longer times for annealing and denaturation are often required.
Moreover, the target sequence may be available in a large or small
amount. Target sequences in smaller amounts are more difficult to
amplify and generally require more PCR reaction time (i.e., more
PCR cycles). Other considerations may also be important depending
on particular uses. For instance, the PCR apparatus may be used to
produce a certain amount of a target sequence for subsequent
applications, experiments, or analyses, or else to detect or
identify a target sequence from a sample. In further
considerations, the PCR apparatus may be used in the laboratory or
in the field, or in certain extraordinary environments, for
instance, inside a car, a ship, a submarine, or a spaceship; under
severe weather conditions, etc.
[0402] As discussed, the thermal convection PCR apparatus of the
present invention generally provides faster and more efficient PCR
amplification than prior PCR apparatuses. Moreover, the invention
apparatus has a substantially lower power requirement and a much
smaller size than prior PCR apparatuses. For instance, the thermal
convection PCR apparatus is typically at least about 1.5 to 2 times
faster (preferably about 3 to 4 times faster) and requires at least
about 5 times (preferably about ten times to several tens of times)
less power for operation with its size or weight at least about 5
to 10 times smaller. Hence, if a suitable design can be selected,
users can have an apparatus that can cost much less time, energy,
and space.
[0403] In order to select a suitable apparatus design, it is
important to appreciate the key functions of an intended
temperature shaping element. As summarized in Table 1 below, each
temperature shaping element has specific functions with regard to
the performance of the thermal convection PCR apparatus. For
instance, the chamber structure generally increases the speed of
the thermal convection within a heat source in which a chamber
resides as compared to the structures without the chamber, and the
thermal brake generally decreases the speed of the thermal
convection as compared to the structures having the chamber
structure without the thermal brake. Importantly, however,
incorporation of the thermal brake structure in addition to the
chamber structure within the second heat source makes the time
length or volume of the sample available for the polymerization
step larger so that efficiency of the PCR amplification can be
increased for target sequences that require a longer polymerization
time. Hence, the chamber structure can be used with or without the
thermal brake depending on particular applications as discussed
below. As also summarized in Table 1, any one or a combination of
the convection accelerating elements (e.g., the positional
asymmetry, the structural asymmetry, and the centrifugal
acceleration) can be used to increase the speed of the thermal
convection regardless of other heat source structures including the
channel alone structure (i.e., a structure without the chamber).
Hence, at least one or a combination of these convection
accelerating elements can be combined with nearly all of the heat
source structures in order to enhance the thermal convection speed
as needed. As discussed, the invention apparatus requires much less
power than prior PCR apparatuses, mainly as a result of eliminating
necessity for the thermal cycling process (i.e., the process that
changes the temperature of the heat source). As also discussed, a
suitable choice of the first insulator (i.e., the thickness of the
insulating gap as well as use of a proper thermal insulator) can
make the power consumption of the invention apparatus further
reduced. Moreover, use of the protrusion structure(s) can still
further reduce the power consumption of the invention apparatus
substantially (see Example 1, for instance) and also to increase
the chamber length and thus to increase the polymerization time.
Other parameters such as the receptor hole depth and the
temperatures of the first and second heat sources can also be used
to modulate the thermal convection speed and also the time period
available for each of the polymerization, annealing and
denaturation steps. As discussed below, each of these temperature
shaping elements can be used alone or in combination with one or
more other elements to construct a particular thermal convection
PCR apparatus that is suitable for a particular application.
TABLE-US-00001 TABLE 1 Key Functions of Temperature Shaping
Elements Temperature Shaping Element Key Functions Chamber
Increases the thermal convection speed within the heat source in
which the chamber resides as compared to the channel alone
structure. The smaller the chamber diameter or the chamber gap, the
slower is the thermal convection speed. Thermal Brake Decreases the
thermal convection speed when combined with the chamber structure.
Typically positioned within the second heat source in combination
with at least one chamber and make the time length and volume of
the sample available for the polymerization step increase as
compared to the chamber only structure. The larger the length of
the thermal brake along the channel axis, the slower is the thermal
convection speed and the larger time and sample volume becomes
available for the polymerization step. Insulator/Insulating gap
Generally required for the multi-stage thermal convection
apparatus. Useful to control the thermal convection speed and to
reduce power consumption. The smaller the length of the insulator
along the channel axis, the larger are the power consumption and
the driving force for the thermal convection. Protrusion Useful to
reduce power consumption substantially and also to lengthen the
chamber length along the channel axis (and thus to increase the
time and sample volume available for the polymerization step).
Positional Asymmetry Increases the thermal convection speed and can
be incorporated into the invention apparatus as an adjustable
structural element so as to provide freedom to control the thermal
convection speed within a given design. When used with a structural
asymmetry, an adjustable positional asymmetry element can be used
as both an accelerating and a decelerating element. Structural
Asymmetry Increases the thermal convection speed. Centrifugal
Acceleration Increases the thermal convection speed while providing
freedom to control the thermal convection speed within a given
design. Typically used with the positional asymmetry.
[0404] Although many useful apparatus embodiments are provided by
the invention, the following combinations are particularly useful
and easy to predict the performance of the invention apparatus.
[0405] An acceptable thermal convection PCR apparatus for many
applications typically includes the channel and the first insulator
(or the first insulating gap) as basic elements. One or more other
temperature shaping elements can be combined to use with these
basic elements. An apparatus that uses the channel and the
insulator only may not be optimal for some PCR applications. With
the channel structure alone, the temperature gradient inside the
sample within each heat source may be too small due to efficient
heat transfer from the heat sources, and thus thermal convection
becomes either too slow or not properly occurring. Use of the
chamber structure can remedy this deficiency. As discussed, the
speed of the thermal convection within each heat source can be
increased by incorporating a chamber structure in that heat source.
Thermal convection PCR apparatuses that use the chamber as an
additional temperature shaping element are generally suited for
most applications including fast amplification of relatively short
target sequences (e.g., shorter than about 1 kbp) having simple
structures as well as longer target sequences (e.g., longer than
about 1 kbp up to about 2 or 3 kbp) or target sequences having
complex structures (e.g., genomic or chromosomal DNAs). For
instance, an apparatus design having a straight chamber in the
second heat source with its width or diameter larger than about 3
or 4 mm can deliver PCR amplification of relatively short sequences
within less than about 20 or 25 min, preferably within less than
about 10 to 15 min depending on the amount and size of the target
sequence (see Example 1, for instance). Amplification of target
sequences having complex structures (e.g., see Example 1 for
amplification of human genome targets) typically takes about 25 or
30 min. Longer target sequences typically takes more time, for
instance, about 30 min to up to about 1 hour depending on the size
and structure of the target sequence. Further increase of the speed
of the thermal convection PCR could be achieved by incorporating at
least one of the convection accelerating elements (e.g., see
Examples 2 and 3).
[0406] Further enhancement of the dynamic range of the thermal
convection PCR apparatus can be achieved by incorporating a thermal
brake and/or a narrower chamber (e.g., smaller than about 3 mm of
the chamber width or diameter) within the second heat source. Use
of a thermal brake or a chamber having a reduced width or diameter
(either partially or completely) within the second heat source
leads to enhanced heat transfer from the second heat source to the
channel, and hence the thermal convection becomes decelerated. In
such decelerated heat source structures, the polymerization time
period can be further increased so as to amplify longer sequences,
for instance, up to about 5 or 6 kbp. However, the total PCR
reaction time could be inevitably increased due to a slow thermal
convection speed, for instance, about 35 min to up to about 1 hour
or longer depending on the size and structure of the target
sequence. Any one or more of the convection accelerating elements
can be combined with this type of apparatus designs to increase the
speed of the thermal convection PCR as desired. In this type of
embodiments, it is typically recommended to use primers having
relatively high melting points (e.g., higher than about 60.degree.
C.) in order to make the temperature of the sample within the
second heat source near or close to the optimum temperature of
typical DNA polymerases.
[0407] The convection accelerating elements mentioned above (i.e.,
the positional asymmetry, the structural asymmetry, and the
centrifugal acceleration) can affect the speed of the thermal
convection in different degrees. The positional or structural
asymmetry can typically enhance the thermal convection speed from
about 10% or 20% up to about 3 to 4 times. In the case of the
centrifugal acceleration, the enhancement can be made as large as
possible, for instance, about 11,200 times at 10,000 rpm when R=10
cm as discussed. A practically useful range would be up to about 10
to about 20 times enhancement. When any one of these convection
accelerating elements is used, the speed of the thermal convection
can be increased. Hence, whenever a further increase of the thermal
convection speed is needed for the user's applications, such
feature can be conveniently incorporated. One particular design
that includes at least one of the convection accelerating elements
is a heat source structure that does not include the chamber (i.e.,
the channel only). Use of a convection accelerating element can
make the channel alone design operable. In such channel alone
embodiment, use of primers having relatively high melting points
(e.g., higher than about 60.degree. C.) is typically recommended in
order to make the temperature of the sample within the second heat
source near or close to the optimum temperature of typical DNA
polymerases. Such channel alone design when used with high melting
point primers is advantageous since it can provide the time period
and volume of the sample available for the polymerization step that
is as largest as possible. However, as discussed, such design
delivers a thermal convection speed that is typically too slow. Use
of any one or more of the convection accelerating elements can
remedy such deficiency by increasing the thermal convection speed
as user's demand.
[0408] All of the apparatus examples discussed above require much
less power than prior PCR apparatuses and can be made as portable
devices, i.e., operable with a battery, even without the protrusion
structure. As discussed, use of the protrusion structure can reduce
the power consumption substantially and thus more recommended if a
portable PCR apparatus is essential for the user's
applications.
[0409] Also, the apparatus designs discussed above can amplify from
very low copy number samples (when optimized). For instance, as
demonstrated in Examples 1 and 2, target sequences even much less
than about 100 copies can be amplified in about 25 min or about 30
min.
[0410] Moreover, the apparatus designs discussed above can be used
in the laboratory or in the field, or in certain extraordinary
conditions, not like many prior PCR apparatuses that can be used
only under controlled conditions such as inside a laboratory. For
instance, we have tested a few invention apparatuses inside a car
while driving and confirmed that fast and efficient PCR
amplification can be achieved as inside a laboratory. Furthermore,
we also tested a few invention apparatuses under extraordinary
temperature conditions (from below about -20.degree. C. to above
about 40.degree. C.) and confirmed fast and efficient PCR
amplification regardless of the outside temperatures.
[0411] Finally, as exemplified throughout the Examples, the thermal
convection PCR apparatuses of the present invention can deliver PCR
amplification that is not only fast but also very efficient. Hence,
it is demonstrated that the invention apparatuses are generally
suitable for nearly all of the diverse different applications of
the PCR apparatus while providing enhanced performance with a new
feature of a palm-size portable PCR device.
[0412] Apparatus with Housing and Temperature Control Elements
[0413] The invention apparatus referred to above can be used alone
or in combination with suitable housing, temperature sensing, and
heating and/or cooling elements. In one embodiment shown in FIG.
30, the first heat source 20 and second heat source 30 features at
least one first securing element 200 (typically a screw hole) and a
second securing element 210 in which each of the elements are
adapted to secure the heat sources and the first insulator 50
together as a single operable unit. The second securing element 210
is preferably "wing-shaped" to help provide a boundary for
additional insulating spaces (see below). Heating and/or cooling
elements 160a and 160b are each positioned in the first 20 and
second 30 heat sources, respectively. Each of the heat sources is
typically equipped with at least one heating element. Typically
useful heating elements are of resistive heating or inductive
heating types. Depending on intended use, one or more of the heat
sources can be further equipped with one or more of cooling
elements and/or one or more of heating elements. Typically
preferred cooling elements are a fan or a Peltier cooler. As well
known, the Peltier cooler can function as both a heating and
cooling element. It is particularly preferred to use more than one
heating elements or both heating and cooling elements in different
locations of one or more of the heat sources when a temperature
gradient operation is required to provide different temperatures
across that heat source. The first 20 and second 30 heat sources
further include temperature sensors 170a and 170b disposed in each
of the heat sources, respectively. For most of the embodiments,
each of the heat sources is typically equipped with one temperature
sensor. However, in some embodiments such as those with a
temperature gradient operation capability in one or more of the
heat sources, two or more temperature sensors can be located at
different positions of that heat source.
[0414] FIGS. 31A-B provide cross-sectional views of the embodiment
shown in FIG. 30. In addition to the cross sectional views of the
channel and chamber structures, locations of the heating and/or
cooling elements are shown as one example. As shown in this
example, it is preferred to position the heating and/or cooling
elements evenly to each of the heat sources to provide a uniform
heating and/or cooling across each of the heat sources. For
instance as depicted in FIG. 31B, the heating and/or cooling
elements are positioned in between each of the channel and chamber
structures and equally spaced from each other (see also FIG. 33 for
instance). The cross-sectional view depicted in FIG. 31A, for
instance, shows connections (i.e., the circles) between the heating
and/or cooling elements from one position in between each of the
channel and chamber structures to another. In other types of
embodiments such as those with a temperature gradient operation
option, two or more of the heating or cooling elements can be used
in one or more of the heat sources and positioned to different
locations of that heat source to provide a biased heating and/or
cooling across that heat source.
[0415] In FIG. 32, the plane of section is through one of the
second securing elements 210 and a first securing element 200. As
shown, the first securing element 200 includes a screw 201, washer
202a, securing element of the first heat source 203a, spacer 202b,
and securing element of the second heat source 203b. Preferably, at
least one of and more preferably all of the screw 201, the washer
202a and the spacer 202b are made from a thermal insulator
material. Examples include plastics, ceramics, and plastic
composites (such as those with carbon or glass fiber). Materials
having a high mechanical strength, high melting and/or deflection
temperature (e.g., about 100.degree. C. or higher, more preferably
about 120.degree. C. or higher), and low thermal conductivity
(e.g., plastics with thermal conductivity smaller than about a few
tenths of Wm.sup.-1l.sup.-1 or ceramics with thermal conductivity
smaller than about a few Wm.sup.-1K.sup.-1) are more preferred.
More specific examples include plastics such as PPS (polyphenylene
sulfide), PEEK (polyetherehterketone), Vesper (polyimide), RENY
(polyamide), etc. or their carbon or glass composites, and low
thermal conductivity ceramics such as Macor, fused silica,
zirconium oxide, Mullite, Accuflect, etc.
[0416] FIG. 33 provides an expanded view of an apparatus embodiment
with various securing element and temperature control elements. It
will be apparent that in addition to the particular securing
structures shown in FIG. 33, others are possible. Thus in one
embodiment, at least one of the first and/or second securing
elements (200, 210) is located in other region(s) of at least one,
and preferably all of the first heat source 20 and second heat
source 30, and first insulator 50. That is, although the second
heat source 30 is shown to include the second securing element 210,
any other or all of the heat sources and/or the first insulator
could include the second securing element 210. In another
embodiment, at least one of the first and/or second securing
elements (200, 210) is located in an inner region of at least one,
and preferably all of the first heat source 20, second heat source
30, and first insulator 50.
[0417] Although the forgoing invention embodiments will be
generally useful for many PCR applications, it will often be
desirable to add protective housing. One embodiment is shown in
FIGS. 34A-B. As shown, the apparatus 10 features a first housing
element 300 that surrounds the first heat source 20, the second
heat source 30, and the first insulator 50. In this embodiment,
each of the second securing elements 210 has a wing-shaped
structure that cooperates with other structural elements of the
apparatus 10 to form at least one insulating gap, for example, one,
two, three, four, five, six, seven or eight of such gaps. Each of
the gaps can be filled with a suitable insulating material such as
those disclosed herein such as a gas or solid insulator. Air will
be a preferred insulating material for many applications. Presence
of the insulating gap(s) provides advantages such as reducing heat
loss from the apparatus 10, thereby lowering power consumption.
[0418] Thus in the embodiment shown in FIG. 34A-B, the second heat
source 30 comprises four second securing elements 210 in which each
pair of second securing elements defines a second insulating gap
310. In particular, FIG. 34A shows four parts of the second
insulating gaps 310 each defined by a first housing element 300 and
a pair of the second securing element 210. FIG. 34A also shows a
third insulating gap 320 located between the bottom of the first
heat source 20 and the first housing element 300. Also shown is a
support 330 for suspending the secured heat sources inside the
first housing element 300, thereby helping form the second
insulating gap 310 and the third insulating gap 320.
[0419] It will often be desirable to further house the invention
apparatus, for example to provide further protection and insulating
gaps. Referring now to FIG. 35A-B, the apparatus further includes a
second housing element 400 that surrounds the first housing element
300. In this embodiment, the apparatus 10 further includes a fourth
insulating gap 410 defined by the first housing element 300 and the
second housing element 400. The apparatus 10 can also include a
fifth insulating gap 420 located between the bottom of the first
housing element 300 and the bottom of the second housing element
400.
[0420] If desired, the invention apparatus may further include at
least one fan unit to remove heat from the apparatus. In one
embodiment, the apparatus comprises a first fan unit positioned
above the second heat source 30 to remove heat from the second heat
source 30. If desired, the apparatus may further include a second
fan unit positioned below the first heat source 20 to remove heat
from the first heat source 20.
Convection PCR Apparatus Incorporating Centrifugal Acceleration
[0421] It is an object of the invention to provide "centrifugal
acceleration" as an optional additional feature of the apparatus
embodiments described herein. As discussed above, it is believed
that thermal convection can be made optimal when a vertical
temperature gradient (and optionally or in addition, a horizontally
asymmetric temperature distribution when the positional or
structural asymmetry is used) is generated inside a fluid.
Proportional to the magnitude of vertical temperature gradient, a
buoyancy force is generated that drives a convection flow inside
the fluid. Thermal convection generated by an invention apparatus
must typically fulfill various conditions for inducing a PCR
reaction. For instance, the thermal convection must flow through a
plurality of spatial regions sequentially and repeatedly, while
maintaining each of the spatial regions at a temperature range
suitable for each step of the PCR reaction (i.e., the denaturation,
annealing, and polymerization steps). Moreover, the thermal
convection must be controlled to have a suitable speed so as to
allow suitable time period for each of the three PCR reaction
steps.
[0422] Without wishing to be bound to any theory, it is believed
that thermal convection can be controlled by controlling the
temperature gradient, more precisely distribution of the
temperature gradient inside the fluid. The temperature gradient
(dT/dS) depends on temperature difference (dT) and distance (dS)
between two reference positions. Therefore, the temperature
difference or distance may be changed to control the temperature
gradient. However, in the convection PCR apparatus, neither the
temperature (or its difference) nor the distance may be changed
easily. The temperature of different spatial regions inside the
sample fluid is subject to a specific range as defined by the
temperature suitable for each of the three PCR reaction steps.
There are not many opportunities to change the temperature of
different (typically at least vertically different) spatial regions
inside the sample. Furthermore, vertical positions of the different
spatial regions (in order to generate a vertical temperature
gradient for inducing a buoyant driving force) are usually
restricted due to a small volume of the sample fluid. For instance,
a typical volume of PCR sample is only about 20 to 50 microliters
and sometimes smaller. Such small volumes and space limitations do
not allow much freedom to change the vertical positions of the
different spatial regions for the PCR reaction.
[0423] As discussed, the buoyancy force is proportional to the
vertical temperature gradient that in turn depends on temperature
difference and distance between two reference points. Further to
such dependence, however, the buoyancy force is also proportional
to the gravitational acceleration (g=9.8 m/sec.sup.2 on Earth).
This force field parameter is a constant, a variable that cannot be
controlled or changed, but can be only defined by the law of
universal gravitation. Therefore, nearly all of the thermal
convection based PCR apparatuses rely upon highly restricted
special structures, inevitably adapted to gravitational forces.
[0424] Use of centrifugal acceleration in accord with the present
invention provides a solution for this problem. By making a
convection based PCR apparatus subject to a centrifugal
acceleration force field, one can control the magnitude of the
buoyant driving force regardless of the structure that defines the
magnitude of the temperature gradient, thereby controlling the
convection speed without much limitation.
[0425] FIGS. 36A-B shows one embodiment of a PCR centrifuge 500
according to the invention. In this example, the apparatus 10 is
attached to a rotation arm 520 rotatably attached to motor 501. In
this embodiment, the rotation arm 520 includes a tilt shaft 530 for
providing freedom of changing the angle between the axis of
rotation 510 and the channel axis 80. The PCR centrifuge may
include any number of the apparatus 10 provided intended results
are achieved, for example, 2, 4, 6, 8, 10 or even 12. The apparatus
10 may or may not include protective housing as discussed above,
although having some protective housing will be generally
useful.
[0426] The tilt shaft 530 is preferably configured to be an angle
inducing element capable of tilting the angle of the heat source
(more particularly the angle of the channel axis 80) with respect
to the rotation axis. Tilt angle can be adjusted depending on the
rotation speed (i.e., depending on the magnitude of the centrifugal
acceleration) so that the tilt angle between the channel axis 80
and the net acceleration vector depicted in FIG. 37 can be adjusted
in the range between from about 0.degree. to about 60.degree.. In
one embodiment, the angle inducing element in FIG. 36A is a
rotation shaft (depicted as a circle) in the center of the joint
region between the horizontal arm and an arm on which the heat
source assembly is located.
[0427] In the embodiment shown in FIGS. 36A-B, the sample fluid
inside the reaction vessel placed inside the apparatus 10 is
subject to a centrifugal acceleration force in addition to the
gravitational acceleration force. See FIG. 37. As will be
appreciated, the direction of the centrifugal acceleration g.sub.c
is perpendicular to (and outward from) the axis of the centrifugal
rotation, and its magnitude is given by an equation g.sub.c=R
.omega..sup.2, where R is the distance from the axis of the
centrifugal rotation to the sample fluid and .omega. is angular
velocity in radian/sec. For instance, when R=10 cm and speed of the
centrifugal rotation is 100 rpm (corresponding to .omega.=about
10.5 radian/sec), magnitude of the centrifugal acceleration is
about 11 m/sec.sup.2, similar to the gravitational acceleration on
Earth. Since the centrifugal acceleration is proportional to square
of the rotation speed (or square of the angular velocity), the
centrifugal acceleration increases quadratically with increase of
the rotation speed, for instance, about 4.5 times of the
gravitational acceleration at 200 rpm, about 112 times at 1,000
rpm, and about 11,200 times at 10,000 rpm when R=10 cm. The
magnitude of the net force field that acts on the sample fluid can
be controlled freely by adopting such centrifugal acceleration.
Therefore, the buoyancy force can be controlled (typically
increased) as needed so as to make the convection speed as fast as
needed. Practically, there are few limitations for inducing the
thermal convection to very high flow speed sufficient for very high
speed PCR reaction, provided a small vertical temperature gradient
can be generated in the sample fluid. Therefore, prior limitations
regarding heat source assembly and use can be minimized or avoided
when combined with centrifugal acceleration in accord with the
invention.
[0428] As depicted in FIG. 37, the sample fluid is subject to the
net force field generated by addition of the centrifugal
acceleration and the gravitational acceleration. In a typical
embodiment, the channel axis 80 is aligned parallel to the net
force field or made to have a tilt angle .theta..sub.c with respect
to the net force field. As discussed, presence of the tilt angle is
generally preferred in order to make the convection flow stay in a
stable route. The tilt angle .theta..sub.c ranges from about
2.degree. to about 60.degree., more preferably about 5.degree. to
about 30.degree..
[0429] It will be appreciated that the apparatus embodiment used to
exemplify the PCR centrifuge 500 is shown in FIGS. 1 and 2A-C.
However, the PCR centrifuge 500 is compatible with use of one or a
combination of different invention apparatuses as described herein.
In particular, the PCR centrifuge 500 can also be used with nearly
any type of heat source structure and reaction vessel described
herein provided that a small vertical temperature gradient can be
generated inside the sample. For example, nearly any of the heat
source structures described above and elsewhere (e.g., WO02/072267
to Benett et al. and U.S. Pat. No. 6,783,993 to Malmquist et al.)
may be combined with the centrifugal element of the present
invention so as to enhance the amplification speed and performance
of the apparatus. Moreover, other heat source structures that
cannot be made operable (or that cannot be made to provide a high
PCR amplification speed) in typical gravitationally driven mode can
be made operable when combined with the centrifugal acceleration
structure. For instance, a heat source structure that does not
include a chamber as described herein but only comprises the
channel structure may also be made operable. See PCT/KR02/01900,
PCT/KR02/01728 and U.S. Pat. No. 7,238,505, for example. In this
embodiment, the prior heat source structures without the chamber
provides a temperature distribution inside the second heat source
that changes slowly, presumably due to a high heat transfer from
the second heat source. A result is a small temperature gradient
within the second heat source. With only gravity, thermal
convection will be unsatisfactory or too slow for many PCR
applications. However, introduction of centrifugal acceleration in
accord with the invention can make thermal convection sufficiently
fast and stable so as to induce the PCR reaction successfully and
efficiently.
[0430] In typical operation of the thermal convection PCR
centrifuge 500, the axis of rotation 510 is essentially parallel to
the direction of gravity. See FIG. 37. In this embodiment, the
channel axis 80 is essentially parallel to, or tilted with respect
to the direction of net force generated by the gravitational force
and the centrifugal force. That is, the channel axis 80 can be
tilted with respect to the direction of net force generated by the
gravitational force and the centrifugal force. For most
embodiments, the tilt angle .theta..sub.c between the channel axis
80 and the direction of the net force is between about 2.degree. to
about 60.degree.. The tilt shaft 530 is adapted to control the
angle between the channel axis 80 and the net force. In operation,
the axis of rotation 510 is usually located outside of the first 20
and second 30 heat sources. Alternatively, the axis of rotation 510
is located essentially at or near the center of the first 20 and
second 30 heat sources. In these embodiments, the apparatus 10
includes a plurality of channels 70 that are located concentrically
with respect to the axis of rotation 510.
[0431] Circular-Shaped Heat Sources
[0432] In another embodiment of the thermal convection PCR
centrifuge, one or more of the heat sources has a circular or
semi-circular shape. FIGS. 38A-B and 39A-B show particular
embodiments of such a heat source structure.
[0433] FIGS. 38A-B show vertical sections of a particular
embodiment of a centrifugally accelerated convection PCR apparatus.
In particular, FIGS. 38A and 38B show cross-sections along the
channel and securing element regions, respectively. The two
sections are defined in FIGS. 39A-B which depict horizontal top
view of the first 20 and second 30 heat sources, respectively. As
depicted in FIGS. 38A-B, the two circular shape heat sources are
assembled to form an apparatus embodiment rotatably attached to the
rotation axis 510 of a PCR centrifuge 500 through a rotation arm
520. The center of the heat source assembly is positioned
concentric with respect to the rotation axis 510 so that the radius
of centrifugal rotation is defined by the horizontal length of the
rotation arm from the rotation axis to the center of the channel
70. The two heat sources 20 and 30 are assembled essentially
parallel to each other with the top of one heat source facing the
bottom of another heat source. As also depicted, the heat source
assembly is oriented with respect to the rotation axis such that
the channel axis 80 is aligned either parallel to, or tilted from
the net acceleration vector depicted in FIG. 37.
[0434] The two heat sources depicted in FIGS. 39A-B are assembled
using a set of first securing element comprising a screw 201,
spacers or washers 202a-b, and securing apertures 203a-b formed in
the heat sources as depicted in FIG. 38B. A second securing element
210 formed in the second heat source 30 shown in FIGS. 38B and 39B
is used to install the apparatus within the first housing element
300.
[0435] Nearly any of the apparatus embodiments disclosed in the
present application (including various channel and chamber
structures) can be used with the centrifugally accelerated thermal
convection PCR apparatus described herein. However, an apparatus
without any chamber structure can also be used.
[0436] In one embodiment of the forgoing thermal convection PCR
centrifuge, the device is made portable and preferably operated
with a battery. The embodiment shown in FIGS. 36A-B can be used for
high throughput large scale PCR amplification, for example. In this
embodiment, the apparatus can be used as a separable module and
thus can be easily loaded and unloaded to the centrifuge unit.
Reaction Vessels
[0437] A suitable channel of the apparatus is adapted to hold a
reaction vessel within the apparatus so that intended results can
be achieved. In most cases, the channel will have a configuration
that is essentially the same as that of a lower part of the
reaction vessel. In this embodiment, the outer profile of the
reaction vessel, particularly the lower part, will be essentially
identical to the vertical and horizontal profiles of the channel.
The upper part of the reaction vessel (i.e., toward the top end)
may have nearly any shape depending on intended use. For instance,
the reaction vessel may have a larger width or diameter on the
upper part to facilitate introduction of a sample and may include a
cap to seal the reaction vessel after introduction of a sample to
be subjected to thermal convection PCR.
[0438] In one embodiment of a suitable reaction vessel, and
referring again to FIG. 7A-D, the outer profile of the reaction
vessel can be identical to the profile of the channel 70 up to the
top end 71 of the channel 70. The shape or profile of inside of the
reaction vessel may have a shape different from that of outside of
the reaction vessel (if wall thickness of the reaction vessel is
made to vary). For instance, the outer profile of the horizontal
section may be circular while the inner profile is ellipsoidal, or
vice versa. Different combinations of outer and inner profiles are
possible as far as the outer profile is suitably selected to
provide proper thermal contact with the heat sources, and the inner
profile is suitably selected for an intended thermal convection
pattern. In typical embodiments, however, the reaction vessel has a
wall thickness that is about constant or does not vary much, i.e.,
the inner profile is typically identical or similar to the outer
profile of the reaction vessel. Typical wall thickness ranges
between from about 0.1 mm to about 0.5 mm, more preferably between
from about 0.2 mm to about 0.4 mm, although it can vary depending
on the material used.
[0439] If desired, the vertical profile of the reaction vessel may
also be shaped to form a linear or tapered tube to fit with the
channel as shown in FIGS. 7A-D. When tapered, the reaction vessel
may be tapered either from the top to the bottom or from the bottom
to the top, although a reaction vessel that is (linearly) tapered
from the top to the bottom is generally preferred as in the case of
the channel. Typical taper angle .theta. of the reaction vessel is
in the range between from about 0.degree. to about 15.degree., more
preferably from about 2.degree. to about 10.degree..
[0440] The bottom end of the reaction vessel may also be made flat,
rounded, or curved as for the bottom end of the channel depicted in
FIGS. 7A-D. When the bottom end is rounded or curved, it can have a
convex or concave shape with its radius of curvature equal to or
larger than the radius or half width of the horizontal profile of
the bottom end. Flat or near flat bottom end is more preferred over
other shapes since it can provide an enhanced heat transfer so as
to facilitate the denaturation process. In such preferred
embodiments, the flat or near flat bottom end has a radius of
curvature that is at least two times larger than the radius or half
width of the horizontal profile of the bottom end.
[0441] Also if desired, horizontal profile of the reaction vessel
may also be made into various different shapes although a shape
having certain symmetry is generally preferred. FIGS. 8A-J shows a
few examples of the horizontal profile of the channel having
certain symmetry. An acceptable reaction vessel may be made to fit
these shapes. For instance, the reaction vessel may have its
horizontal shape that is circular (top, left), square (middle,
left), or rounded square (bottom, left) generally the same as that
shown for the channel 70 in FIGS. 8A, D, G, and J. Thus, the
reaction vessel may have a horizontal shape that has its width
larger than its length (or vice versa), for instance, an ellipsoid
(top, middle), rectangular (middle, middle), or rounded rectangular
(bottom, middle) that is generally the same as that depicted in the
middle column of FIGS. 8B, E, and H for the channel 70. This type
of horizontal shape for the reaction vessel is useful when
incorporating a convection flow pattern going upward on one side
(e.g., on the left hand side) and going downward on the opposite
side (e.g., on the right hand side). Due to the relatively larger
width profile incorporated compared to the length, interference
between the upward and downward convection flows can be reduced,
leading to more smooth circulative flow. The reaction vessel may
have a horizontal shape that has its one side narrower than the
opposite side. A few examples are shown on the right column of
FIGS. 8A-J for the shape of the channel. In particular, the
reaction vessel may be made so that the left side of the reaction
vessel is narrower than the right side for instance, as shown in
FIGS. 8C, F and I for the channel 70. This type of horizontal shape
is also useful when incorporating a convection flow pattern going
upward on one side (e.g., on the left hand side) and going downward
on the opposite side (e.g., on the right hand side). Moreover, when
this type of shape is incorporated, speed of the downward flow
(e.g., on the right hand side) can be controlled (typically
reduced) with respect to the upward flow. Since the convective flow
must be continuous within the continuous medium of the sample, the
flow speed should be reduced when cross-sectional area becomes
larger (or vice versa). This feature is particularly important with
regard to enhancing the polymerization efficiency. The
polymerization step typically takes place during the downward flow
(i.e., after the annealing step), and therefore time period for the
polymerization step can be lengthened by making the downward flow
slower as compared to that of the upward flow, leading to more
efficient PCR amplification.
[0442] Further examples of suitable reaction vessels are provided
in FIGS. 40A-D. As shown, the reaction vessel 90 includes a top end
91 and a bottom end 92 which ends include center points that define
a central reaction vessel axis 95. The reaction vessel 90 is
further defined by an outer wall 93 and an inner wall 94 which
surround a region for holding a PCR reaction mixture. In FIGS.
40A-B, the reaction vessel 90 is tapered from the top end 91 to the
bottom end 92. A generally useful taper angle (.theta.) is in the
range between from about 0.degree. to about 15.degree., preferably
from about 2.degree. to about 10.degree.. In the embodiment shown
in FIG. 40A, the reaction vessel 90 has a flat or near flat bottom
end 92 while in the example shown in FIG. 40B, the bottom end is
curved or rounded. The top 71 and bottom 72 ends of the channel are
marked in FIGS. 40A-D.
[0443] FIGS. 40C-D provide examples of suitable reaction vessels
with straight walls from the top end 91 to the bottom end 92. The
reaction vessel 90 shown in FIG. 40C has a flat or near flat bottom
end 92 while in the example shown in FIG. 40D, the bottom end is
curved or rounded.
[0444] Preferably, the vertical aspect ratio of the outer wall 93
of the reaction vessel 90 shown in FIGS. 40A-D is at least about 4
to about 15, preferably from about 5 to about 10. The horizontal
aspect ratio of the reaction vessel is defined by the ratio of the
height (h) to the width (w1) up to the position corresponding to
the top end 71 of the channel 70 as in the case of the channel. The
horizontal aspect ratio of the outer wall 93 is typically about 1
to about 4. The horizontal aspect ratio is defined by the ratio of
the first width (w1) to the second width (w2) of the reaction
vessel along first and second directions, respectively, that are
mutually perpendicular to each other and aligned perpendicular to
the channel axis. Preferably, the height of the reaction vessel 90
along the reaction vessel axis 95 is at least between about 6 mm to
about 35 mm. In this embodiment, the average of the width of the
outer wall is between about 1 mm to about 5 mm, and that of the
inner wall of the reaction vessel is between about 0.5 mm to about
4.5 mm.
[0445] FIGS. 41A-J show horizontal cross-sectional views of
suitable reaction vessels for use with the invention. The invention
is compatible with other reaction vessel configurations provided
intended results are achieved. Accordingly, the horizontal shape of
an acceptable reaction vessel can be one or a combination of
circle, semi-circle, rhombus, square, rounded square, ellipsoidal,
rhomboid, rectangular, rounded rectangular, oval, triangular,
rounded triangular, trapezoidal, rounded trapezoidal or oblong
shape. In many embodiments, the inner wall is disposed essentially
symmetrically with respect to the reaction vessel axis. For
example, the thickness of the reaction vessel wall can be between
about 0.1 mm to about 0.5 mm. Preferably, the thickness of the
reaction vessel wall is essentially unchanged along the reaction
vessel axis 95.
[0446] In one embodiment of the reaction vessel 90, the inner wall
94 is disposed off-centered with respect to the reaction vessel
axis 95. For instance, the thickness of the reaction vessel wall is
between about 0.1 mm to about 1 mm. Preferably, the thickness of
the reaction vessel wall is thinner on one side than the other side
by at least about 0.05 or 0.1 mm.
[0447] As discussed, bottom end of a suitable reaction vessel can
be flat, curved or rounded. In one embodiment, the bottom end is
disposed essentially symmetrically with respect to the reaction
vessel axis. In another embodiment, the bottom end is disposed
asymmetrically with respect to the reaction vessel axis. The bottom
end may be closed and can include or consist of a plastic, ceramic
or a glass. For some reactions, the reaction vessel may further
include an immobilized DNA polymerase. Nearly any reaction vessel
described herein can include a cap in sealing contact with the
reaction vessel.
[0448] In embodiments where a reaction vessel is used with a
thermal convection PCR centrifuge of the invention, relatively
large forces will be generated by centrifugal rotation. Preferably,
the channel and the reaction vessel will have a smaller diameter or
width thus having a large vertical profile can be used. The
diameter or width of the channel and the outer wall of the reaction
vessel is at least about 0.4 mm to up to about 4 to 5 mm, and that
of the inner wall of the reaction vessel is at least about 0.1 mm
to up to about 3.5 to 4.5 mm.
Convection PCR Apparatus Comprising an Optical Detection Unit
[0449] It is objective of the invention to provide "optical
detection" as an additional feature of the apparatus embodiments
described herein. It is important to detect progress or results of
the polymerase chain reaction (PCR) during or after the PCR
reaction with speed and accuracy. The optical detection feature can
be useful for such needs by providing apparatuses and methods for
simultaneous amplification and detection of the PCR reaction.
[0450] In typical embodiments, a detectable probe that can
generates an optical signal as a function of the amount of the
amplified PCR product is introduced to the sample, and the optical
signal from the detectable probe is monitored or detected during or
after the PCR reaction without opening the reaction vessel. The
detectable probe is typically a detectable DNA binding agent that
changes its optical property depending on its binding or
non-binding to DNA molecules or interaction with the PCR reaction
and/or the PCR product. Useful examples of the detectable probe
include, but not limited to, intercalating dyes having a property
of binding to double-stranded DNA and various oligonucleotide
probes having detectable label(s).
[0451] The detectable probe that can be used with the invention
typically changes its fluorescence property such as its
fluorescence intensity, wavelength or polarization, depending on
the PCR amplification. For instance, intercalating dyes such as
SYBR green 1, YO-PRO 1, ethidium bromide, and similar dyes generate
fluorescence signal that is enhanced or activated when the dye
binds to double-stranded DNA. Hence, fluorescence signal from such
intercalating dyes can be detected to monitor the amount of the
amplified PCR product. Detection using the intercalating dye is
non-specific with regard to the sequence of the double-stranded
DNA. Various oligonucleotide probes that can be used with the
invention are known in the field. Such oligonucleotide probes
typically have at least one detectable label and a nucleic acid
sequence that can specifically hybridize to the amplified PCR
product or the template. Hence, sequence-specific detection of the
amplified PCR product, including allelic discrimination, is
possible. The oligonucleotide probes are typically labeled with an
interactive label pair such as a pair of two fluorescers or a pair
of a fluorescer and a quencher whose interaction (such as
"fluorescent resonance energy transfer" or "non-fluorescent energy
transfer") is enhanced as the distance between the two labels
becomes shorter. Most of the oligonucleotide probes are designed
such that the distance between the two interactive labels is
modulated depending on its binding (typically a longer distance) or
non-binding (typically a shorter distance) to a target DNA
sequence. Such hybridization-dependent distance modulation results
in change of the fluorescence intensity or change (increase or
decrease) of the fluorescence wavelength depending on the amount of
the amplified PCR product. In other types of the oligonucleotide
probes, the probes are designed to undergo certain chemical
reactions during the extension step of the PCR reaction, such as
hydrolysis of the fluorescer label due to the 5'-3' nuclease
activity of a DNA polymerase or extension of the probe sequence.
Such PCR reaction dependent changes of the probes lead to
activation or enhancement of a fluorescence signal from the
fluorescer so as to signal the change of the amount of the PCR
product.
[0452] A variety of suitable detectable probes and devices for
detecting such probes are described in the following U.S. Pat. Nos.
5,210,015; 5,487,972; 5,538,838; 5,716,784; 5,804,375; 5,925,517;
5,994,056; 5,475,610; 5,602,756; 6,028,190; 6,030,787; 6,103,476;
6,150,097; 6,171,785; 6,174,670; 6,258,569; 6,326,145; 6,365,729;
6,703,236; 6,814,934; 7,238,517, 7,504,241; 7,537,377; as well as
non-US counterpart applications and patents.
[0453] As used herein, the phrase "optical detection unit"
including plural forms means a device(s) for detecting PCR
amplification that is compatible with one or more of the PCR
thermal convection apparatuses and PCR methods disclosed herein. A
preferred optical detection unit is configured to detect a
fluorescence optical signal such as when a PCR amplification
reaction is in progress. Typically, the device will provide for
detection of the signal and preferably quantification thereof
without opening at least one reaction vessel of the apparatus to
which it is operably attached. If desired, the optical detection
unit and one or more of the PCR thermal convection apparatuses of
the invention can be configured to relate the amount of amplified
nucleic acid in the reaction vessel (i.e., real-time or
quantitative PCR amplification). A typical optical detection unit
for use with the invention includes one or more of the following
components in an operable combination: an appropriate light
source(s), lenses, filters, mirrors, and beam-splitter(s) for
detecting fluorescence typically in the visible region between from
about 400 to about 750 nm. A preferred optical detection unit is
positioned below, above and/or to the side of a reaction vessel
sufficient to receive and output light for detecting PCR
amplification within the reaction vessel.
[0454] An optical detection unit is compatible with a thermal
convection PCR apparatus of the invention if it supports robust,
sensitive and rapid detection of the PCR amplification for which
the apparatus is intended. In one embodiment, the thermal
convection PCR apparatus includes an optical detection unit that
enables detection of an optical property of the sample in the
reaction vessel. The optical property to be detected is preferably
fluorescence at one or more wavelengths depending on the detectable
probe used, although absorbance of the sample is sometimes useful
to detect. When fluorescence from the sample is detected, the
optical detection unit irradiates the sample (either a portion of,
or entire sample) with an excitation light and detects a
fluorescence signal from the sample. The wavelength of the
excitation light is typically shorter than the fluorescence light.
In the case of detecting absorbance, the optical detection unit
irradiates the sample with a light (typically at a selected
wavelength or with scanning the wavelength) and the intensity of
the light before and after passing through the sample is measured.
Fluorescence detection is generally preferred because it is more
sensitive and specific to the target molecule to be detected.
[0455] Reference to the following figures and descriptions is
intended to provide greater understanding of the thermal convection
PCR apparatus comprising an optical detection unit for fluorescence
detection. It is not intended and should not be read as limiting
the scope of the present invention.
[0456] Referring to FIGS. 59A-B, the apparatus embodiments feature
one or more optical detection units 600-603 operable to detect a
fluorescence signal from the sample in the reaction vessel 90 from
the bottom end 92 of the reaction vessel 90 or the bottom end 72 of
the channel 70. Shown in FIG. 59A is an embodiment in which single
optical detection unit 600 is used to detect fluorescence from
multiple reaction vessels 90. In this embodiment, a broad
excitation beam (shown as upward arrows) is generated to irradiate
multiple reaction vessels and a fluorescence signal (shown as
downward arrows) from multiple reaction vessels 90 is detected. In
this embodiment, a detector 650 (see FIG. 62, for instance) to be
used for the fluorescence detection is preferably one that has an
imaging capability so that the fluorescence signal from different
reaction vessels can be distinguished from the fluorescence image.
Alternatively, multiple detectors 650 each of which detects the
fluorescence signal from each reaction vessel can be
incorporated.
[0457] In the embodiment shown in FIG. 59B, multiple optical
detection units 601-603 are incorporated. In this embodiment, each
optical detection unit irradiates the sample in each reaction
vessel 90 with an excitation light and detects a fluorescence
signal from each sample. This embodiment is advantageous in
controlling the profile of the excitation beam for each reaction
vessel more precisely and also measuring different fluorescence
signal from different reaction vessels independently and
simultaneously. This type of embodiment is also advantageous in
constructing miniaturized apparatuses since larger optical elements
and greater optical paths required for generating a broad
excitation beam in the single optical detection unit embodiment can
be avoided.
[0458] Again referring to FIGS. 59A-B, when the optical detection
unit 600-603 is located on the bottom end 92 of the reaction vessel
90, the first heat source 20 comprises an optical port 610 for each
channel 70 to provide a path for the excitation and emission light
to the reaction vessel 70. The optical port 610 may be a through
hole or an optical element made of (partially or entirely) an
optically transparent or semitransparent material such as glass,
quartz or polymer materials having such optical property. If the
optical port 610 is made as a though hole, the diameter or width of
the optical port is typically smaller than that of the bottom end
72 of the channel 70 or the bottom end 92 of the reaction vessel
90. In the embodiments shown in FIGS. 59A-B, the bottom end 92 of
the reaction vessel 90 also works as an optical port. Therefore, it
is generally desirable to have all or at least the bottom end 92 of
the reaction vessel 90 made of an optically transparent or
semitransparent material.
[0459] Turning now to FIGS. 60A-B, the apparatus embodiments
feature single optical detection unit 600 (FIG. 60A) or multiple
optical detection units 601-603 (FIG. 60B) that are located above
the top end 91 of the reaction vessel 90. Again, when a single
optical detection unit 600 is incorporated (FIG. 60A), a broad
excitation beam (shown as downward arrows) is generated to
irradiate the multiple reaction vessels and a fluorescence signal
(shown as upward arrows) from the multiple reaction vessels 90 is
detected. When multiple optical detection units 601-603 (FIG. 60B)
are incorporated, each optical detection unit irradiates the sample
in each reaction vessel 90 with an excitation light and detects a
fluorescence signal from each sample.
[0460] In the embodiments shown in FIGS. 60A-B, a center part of a
reaction vessel cap (not shown) that typically fits to the top end
(opening) 91 of the reaction vessel 90 functions as an optical port
for the excitation and emission light. Therefore, all or at least
the center part of the reaction vessel cap is made of an optically
transparent or semitransparent material.
[0461] FIG. 61 shows an apparatus embodiment that features optical
detection units 600 that are located on the side of the reaction
vessel 90. In this particular embodiment, the optical port 610 is
formed on the side of the second heat source 30 (shown as gray
rectangular boxes) and the side of the first insulator 50 (shown as
dashed lines). Alternatively, the optical port 610 can be formed
any one or more of the first 20 and second 30 heat sources, and the
first insulator 50 depending on the position of the fluorescence
detection as required by particular application purposes. In this
embodiment, a side part of the reaction vessel 90 and a portion of
the first chamber 100 along the light path also function as optical
port, and thus all or at least the parts of the reaction vessel 90
and the first chamber 100 are made of an optically transparent or
semitransparent material. When the optical detection unit 600 is
located on the side of the reaction vessel 90, the channels 90 are
typically formed in one or two arrays that are linearly or
circularly arranged. Such arrangement of the channels 70 enables to
detect a fluorescence signal from every channel 70 or reaction
vessel 90 without interference by other channels.
[0462] In the embodiments described above, both excitation and
fluorescence detection are performed from the same side with
respect to the reaction vessel 90, and thus both an excitation part
and a fluorescence detection part are located on the same side,
typically within a same compartment of an optical detection unit
600-603. For instance, in the embodiments shown in FIGS. 59A-B, the
optical detection unit 600-603 that contains both parts is located
on the bottom end 92 of the reaction vessel 90. Similarly, entire
optical detection unit is located above the top end 91 of the
reaction vessel 90 in the embodiments shown in FIGS. 60A-B, and on
the side part of the reaction vessel 90 in the embodiment shown in
FIG. 61. Alternatively, the optical detection unit 600-603 may be
modified so that the excitation part and the fluorescence detection
part are located separately. For instance, the excitation part is
located on the bottom (or top) of the reaction vessel 90 and the
fluorescence detection part is located on the top (bottom) or side
part of the reaction vessel 90. In other embodiments, the
excitation part may be located on one side (e.g., left side) of the
reaction vessel 90 and the fluorescence detection part may be
located another side (e.g., top, bottom, right, front or back side;
or a side part other than the excitation side).
[0463] The optical detection unit 600-603 typically comprises an
excitation part that generates an excitation light with a selected
wavelength and a fluorescence detection part that detects a
fluorescence signal from the sample in the reaction vessel 90. The
excitation part typically comprises a combination of light sources,
wavelength selection elements, and/or beam shaping elements.
Examples of the light source include, but not limited to, arc lamps
such as mercury arc lamps, xenon arc lamps, and metal-halide arc
lamps, lasers, and light-emitting diodes (LED). The arc lamps
typically generate multiple bands or broad bands of light, and the
lasers and LEDs typically generate a monochromatic light or a
narrow band light. The wavelength selection element is used to
select an excitation wavelength from the light generated by the
light source. Examples of the wavelength selection element includes
a grating or a prism (for dispersing the light) combined with a
slit or an aperture (for selecting a wavelength), and an optical
filter (for transmitting a selected wavelength). The optical filter
is generally preferred because it can effectively select specific
wavelength with compact size and it is relatively cheap. Preferred
optical filter is an interference filter having a thin-film coating
that can transmit certain band of light (band-pass filter) or light
having wavelength longer (long-pass filter) or shorter (short-pass
filter) than certain cut-on value. Acoustic optical filters and
liquid crystal tunable filters can be an excellent wavelength
selection element since these types of filters can be
electronically controlled to change the transmission wavelength
with speed and accuracy in a compact size although relatively
expensive. A colored filter glass can also be used as a wavelength
selection element as a cheap replacement of, or in combination with
other types of the wavelength selection element to enhance
rejection of undesired light (e.g., IR, UV, or other stray light).
Choice of the optical filter depends on the characteristics of the
light generated by the light source and the wavelength of the
excitation light as well as other geometric requirement of the
apparatus such as the size. The beam shaping element is used to
shape and guide the excitation beam. The beam shaping element can
be any one or combination of lenses (convex or concave), mirrors
(convex, concave, or elliptical), and prisms.
[0464] The fluorescence detection part typically comprises a
combination of detectors, wavelength selection elements, and/or
beam shaping elements. Examples of the detector include, but not
limited to, photomultiplier tubes (PMT), photodiodes,
charge-coupled devices (CCD), and video camera. The photomultiplier
tubes are typically most sensitive. Therefore, when the sensitivity
is the key issue due to very weak fluorescence signal, the
photomultiplier tube can be a suitable choice. However, the
photomultiplier tubes are not suitable if a compact size or an
imaging capability is required (due to its large size). CCDs,
silicon photodiodes, or video cameras intensified with, for
example, a microchannel plate can have sensitivity similar to the
photomultiplier tubes. If imaging of the fluorescence signal is not
required and miniaturization is important as in the embodiments
having an optical detection unit for each reaction vessel,
photodiodes or CCDs with or without an intensifier can be a good
choice since these elements are compact and relatively cheap. If
imaging is required as in the embodiments having single optical
detection unit for multiple reaction vessels, CCD arrays,
photodiode arrays, or video cameras (also with or without an
intensifier) can be incorporated. Similar to the excitation part,
the wavelength selection element is used to select an emission
wavelength from the light collected from the sample and the beam
shaping element is used to shape and guide the emission light for
efficient detection. Examples of the wavelength selection element
and the beam shaping element are the same as those described for
the excitation part.
[0465] In addition to the optical elements described above, the
optical detection unit can comprise a beam-splitter. The
beam-splitter is particularly useful if the excitation part and the
fluorescence detection part are located on the same side with
respect to the reaction vessel 90. In such embodiments, the paths
of the excitation and emission beams (along opposite directions)
coincide with each other and thus it becomes necessary to separate
the beam paths using a beam-splitter. Typically useful
beam-splitters are dichroic beam-splitters or dichroic mirrors that
have a thin-film interference coating similar to the thin-film
optical filters. Typical beam-splitters reflect the excitation
light and transmit the fluorescence light (a long-pass type), or
vice versa (a short-pass type).
[0466] Referring now to FIGS. 62-63, 64A-B, and 65, a few design
examples of structure of the optical detection unit 600 are
described.
[0467] In FIG. 62, one embodiment of the optical detection unit 600
is illustrated. In this embodiment, excitation optical elements
(620, 630, and 640) are located along a direction at a right angle
with respect to the channel axis 80, and fluorescence detection
optical elements (650, 655, 660, and 670) are located along the
channel axis 80. A dichrocic beam-splitter 680 that transmits the
fluorescence emission and reflects the excitation light (i.e., a
long-pass type) is located around the middle. As typical, a light
generated by the light source 620 is collected by an excitation
lens 630 and filtered with an excitation filter 640 to select an
excitation light with a desired wavelength. The selected excitation
light is then reflected by a dichroic beam-splitter and irradiated
to the sample. Fluorescence emission from the sample is collected
by an emission lens 660 after passing through the dichroic
beam-splitter 680 and an emission filter 670 to select an emission
light with a desired wavelength. The fluorescence light thus
collected is then focused to an aperture or slit 655 or to a
detector 650 to measure the fluorescence signal. The function of
the aperture or slit 655 is "spatial filtering" of the emission.
Typically, the fluorescence light is focused on or near the
aperture or slit 655 and thus a fluorescence image from certain
(vertical) location of the sample is formed on the aperture or slit
655. Such optical arrangement enables to collect a fluorescence
signal efficiently from a certain limited location inside the
sample (e.g., the annealing, extension or denaturation region)
while rejecting light from other locations. Use of the aperture or
slit 655 is optional depending on the type of the detectable probe
used. If the fluorescence signal is subject to be generated from a
specific region inside the sample, use of one or more of the
aperture or slit 655 is preferred. If the fluorescence signal is
subject to be generated regardless of the location inside the
sample, use of the aperture or slit 655 may not be necessary or one
having a larger opening may be used.
[0468] As shown in the embodiment depicted in FIG. 63, the optical
detection unit 600 may be modified to position the excitation
optical elements (620, 630, 640) along the channel axis 80 and the
fluorescence detection optical elements (650, 655, 660, and 670)
along a direction at a right angle to the channel axis 80. A
dichrocic beam-splitter 680 useful for this type of embodiment is a
short-pass type that transmits the excitation light and reflects
the emission light.
[0469] The excitation lens 630 used in the embodiments shown in
FIGS. 62-63 can be replaced with a combination of more than one
lenses or a combination of lenses and mirrors. When a combination
of such optical elements is used, the first lens (typically a
convex lens) is preferably located close to and in front of the
light source in order to collect the excitation light efficiently.
To further enhance the collection efficiency of the excitation
light, a mirror (typically concave or elliptic) may be placed on
the back side of the light source. When it is required to make the
excitation beam large as in the embodiments having a single optical
detection unit 600 for irradiating multiple reaction vessels 90, a
concave lens or a convex mirror may be used additionally to expand
the excitation beam. In some embodiments, one or more of the
optical elements (e.g., one or more of lenses or mirrors) may be
placed other locations, e.g., between the reaction vessel 90 and
the dichroic beam-splitter 680 or the excitation filter 640. In
other aspect, the excitation light is typically shaped to an
essentially collinear beam so as to irradiate a larger volume of
the sample(s). In some special applications such as when using a
multi-photon excitation scheme, the excitation light may be tightly
focused to a certain position inside the sample.
[0470] The emission lens 660 used in the embodiments shown in FIGS.
62-63 can also be replaced with a combination of more than one
lenses or a combination of lenses and mirrors. When a combination
of such optical elements is used, the first lens (typically a
convex lens) is preferably located close to the reaction vessel 90
(for instance, between the reaction vessel 90 and the dichroic
beam-splitter 680 or the emission filter 670) in order to collect
the fluorescence light more efficiently. In some embodiments, one
or more of the optical elements (e.g., a lens or a mirror) may be
placed other locations, e.g., between the reaction vessel 90 and
the dichroic beam-splitter 680 or the emission filter 670.
[0471] FIGS. 64A-B show embodiments in which one lens 635 is used
to shape both the excitation beam and the emission beam. Two
examples of arranging the excitation optical elements (620 and 640)
and the fluorescence detection optical elements (650, 655, and 670)
are shown. The excitation optical elements (620 and 640) are
located along a direction at a right angle to the channel axis 80
in FIG. 64A and along the channel axis 80 in FIG. 64B. This type of
embodiments using a single lens is useful in miniaturizing the
optical detection unit 600 such as in the embodiments of
incorporating multiple optical detection units shown in FIGS. 59B,
60B and 61.
[0472] FIG. 65 shows one apparatus embodiment in which the optical
detection unit 600 is located on the top side of the reaction
vessel 90. The arrangement of the optical elements depicted is the
same as the embodiment shown in FIG. 62. Other types of the optical
arrangements (e.g., those shown FIGS. 63 and 64A-B) can also be
incorporated. When the optical detection unit 600 (or the
excitation or fluorescence detection part) is located on the top
side of the reaction vessel 90, the center part of the reaction
vessel cap 690 functions as an optical port 610. Therefore, as
discussed, the reaction vessel cap 690 or at least the center part
is preferably made of an optically transparent or semitransparent
material in this type of embodiments.
[0473] Again referring to FIG. 65, the reaction vessel 90 and the
reaction vessel cap 690 typically has a sealing relationship with
each other in order to avoid an evaporative loss of the sample
during the PCR reaction. In the reaction vessel embodiment shown in
FIG. 65, the sealing relationship is made between an inner wall of
the reaction vessel 90 and an outer wall of the reaction vessel cap
690. Alternatively, the sealing relationship may be made between an
outer wall of the reaction vessel 90 and an inner wall of the
reaction vessel cap 690 or between a top surface of the reaction
vessel 90 and a bottom surface of the reaction vessel cap 690. In
some embodiments, the reaction vessel cap 690 may be a thin-film
adhesive tape that is optically transparent or semitransparent. In
such embodiments, the sealing relationship is made between a top
surface of the reaction vessel 90 and a bottom surface of the
reaction vessel cap 690.
[0474] The reaction vessel embodiments described above may not be
optimal for all uses of the invention. For instance, and as shown
in FIG. 65, it is typical that the sample meniscus (i.e., a
water-air interface) is formed between the sample and the reaction
vessel cap 690 (or an optical port part of the reaction vessel cap
690). In operation, water in the sample evaporates and condenses to
the inner surface of the reaction vessel cap 690 (or an optical
port part of the reaction vessel cap 690) due to the PCR reaction
that involves a high temperature process. Such condensed water may,
for some applications, interfere somewhat with the excitation beam
and the fluorescence beam, particularly when the optical detection
unit is positioned on the top side of the reaction vessel 90.
[0475] The reaction vessel embodiments exemplified in FIGS. 66A-B
provide another approach. As shown, a reaction vessel 90 and a
reaction vessel cap 690 are designed to have an optical port 695 to
contact the sample. A sample meniscus is formed higher than, or
about the same height as the bottom surface 696 of the optical port
695. Unlike the typical reaction vessel embodiments described
above, the excitation beam and the fluorescence beam are
transmitted directly from the optical port 695 to the sample or
vice versa without passing through the air or any condensed water
inside the reaction vessel 90. Structural requirements for such
embodiments are as follows:
[0476] Firstly, as shown FIGS. 66A-B, the reaction vessel cap 690
has a sealing relationship with the upper part of the reaction
vessel 90 and also with the optical port 695. As discussed, the
sealing between the reaction vessel 90 and the reaction vessel cap
690 can be made at an inner wall of the reaction vessel (as in
FIGS. 66A-B) or at an outer wall or a top end 91 of the reaction
vessel 90. The sealing between the reaction vessel cap 690 and the
optical port 695 can be made at a top surface 697 (FIG. 66A) or a
side wall 699 (FIG. 66B) of the optical port 695. Alternatively the
reaction vessel cap 690 and the optical port 695 may be made as one
body, preferably using a same or similar optically transparent or
semitransparent material.
[0477] Additionally, the diameter or width of the optical port 695
(and also that of a wall of the reaction vessel cap 690 if that
wall is located near or about the same height as the bottom surface
696 of the optical port 695) is made smaller than the diameter or
width of a portion of the inner wall of the reaction vessel 90 that
is located near or about the same height as the bottom surface 696
of the optical port 695. Moreover, the bottom surface 696 of the
optical port 695 is located lower than, or about the same height as
the bottom of the inner part of the reaction vessel cap 690. When
these structural requirements are met, an open space 698 is
provided between the inner wall of the reaction vessel 90 and the
side part of the optical port 695. Therefore, the sample can fill
up a portion of the open space to form a sample meniscus above the
bottom part 696 of the optical port 695 when the reaction vessel 90
is sealed with the reaction vessel cap 690 to make the bottom of
the optical port contact the sample.
[0478] In FIG. 67, use of the optically non-interfering reaction
vessel discussed above is exemplified. As discussed, the bottom 696
of the optical port 695 contacts the sample and the sample meniscus
is formed above the bottom 696 of the optical port 695. With an
optical detection unit 600 located on the top end 91 of the
reaction vessel 90, the excitation beam and the fluorescence beam
are transmitted directly from the optical port 695 to the sample or
vice versa without passing through the air or any condensed water
inside the reaction vessel 90. Such optical structure can greatly
facilitate the optical detection feature of the invention.
Convection PCR Apparatus Comprising a Nucleic Acid Separation
Unit
[0479] It is a further object of the invention to provide at least
one "nucleic acid separation" unit operably linked to the
multi-stage thermal convection apparatus invention described herein
(e.g., one, two, three or more of such units). As will be
appreciated, it will often be important to separate the PCR
amplified product(s) produced by the apparatus during or after the
PCR reaction. In such embodiments, the additional feature of having
the operably linked nucleic acid separation unit will assist
identification, analysis and/or utilization of the amplified PCR
product. Preferably, the nucleic acid separation can be performed
as a function of size or size to charge ratio and/or in combination
with optional optical detection of the separated product(s). The
nucleic acid separation feature can be useful in embodiments that
require simultaneous amplification and separation as well as
identification of the PCR product(s).
[0480] In one embodiment, the multi-stage thermal convection PCR
apparatus is a two-stage apparatus as described herein that
includes an operably linked nucleic acid separation unit that can
separate the amplified PCR product(s). Preferably, the nucleic acid
separation unit separates the PCR product(s) as a function of size
or size to charge ratio. Examples of the size-dependent nucleic
acid separation unit include, but not limited to, a capillary
electrophoresis unit, a gel electrophoresis unit, and other types
of electrophoresis or chromatography units known in the field.
[0481] In another embodiment, the multi-stage thermal convection
PCR apparatus is a two-stage apparatus as described herein that
further comprises at least one operably linked optical detection
unit for detecting the separated PCR product (e.g., one, two, three
or more of such units). For most applications, the optical
detection unit typically detects fluorescence, absorbance, or
chemiluminescence from the PCR product as a function of elution
time and/or as a function of position within the separation
unit.
[0482] Examples of suitable nucleic acid separation units and/or
optical detection units include, but not limited to those described
in the following references: U.S. Pat. Nos. 4,865,707; 5,147,517;
5,384,024; 5,582,705; 5,597,468; 5,790,727; 6,017,434; and
7,361,259; as well as non-US counterpart applications and patents.
See also Felhofer, J. L., et al., Electrophoresis, 31(15), pp.
2469-2486 (2010); Terabe, S., et al., Analytical Chemistry, 56, pp.
111-113 (1984); Jorgenson, J. W. and Lukacs, K. D., Science, 222,
pp. 266-272 (1983); Hjerten, S., Journal of Chromatography 270, pp.
1-6 (1983); and Jorgenson, J. W. and Lukacs, K. D., Analytical
Chemistry, 53(8), pp. 1298-1302 (1981).
[0483] In one embodiment in which the two-stage apparatus includes
an operably linked optical detection unit, at least one detectable
probe (e.g., one, two, three or more of such probes) that can
generate an optical signal as a function of the amount of the PCR
product is introduced to the sample during or after the PCR
reaction, and the optical signal from the detectable probe is
monitored or detected during or after the nucleic acid separation.
The detectable probe is typically a detectable label that generates
a fluorescence, absorbance or chemiluminescence signal, or a
detectable DNA binding agent that generates an optical signal or
changes its optical property depending on its binding or
non-binding to, or interaction with the PCR product. Useful
examples of the detectable probe include, but not limited to,
detectable labels that can be incorporated into the primers or PCR
products, intercalating dyes having a property of binding to
double-stranded DNA, and various oligonucleotide probes having
detectable label(s). Suitable detectable probes include, but are
not limited to the following U.S. Pat. Nos. 5,210,015; 5,487,972;
5,538,838; 5,716,784; 5,804,375; 5,925,517; 5,994,056; 5,475,610;
5,602,756; 6,028,190; 6,030,787; 6,103,476; 6,150,097; 6,171,785;
6,174,670; 6,258,569; 6,326,145; 6,365,729; 6,703,236; 6,814,934;
7,238,517; 7,504,241; and 7,537,377; as well as non-US counterpart
applications and patents.
[0484] The optical detection unit may be used to determine the size
of one or more of the PCR products or in some embodiments to
determine a partial or whole nucleic acid sequence of the PCR
product. When the sequence of the PCR product is to be determined,
the PCR reaction may be terminated by adding a termination agent
such as dideoxynucleotide triphosphates (ddNTPs).
[0485] Thus in a particular invention embodiment, the multi-stage
thermal convention apparatus is a two-stage apparatus as described
herein that further includes as operably linked components, a
suitable nucleic acid separation unit and an optical detection
unit. In use, the two-stage apparatus with the operably linked
nucleic acid separation and optical detection units may be used in
conjunction with an appropriate detectable probe for monitoring or
detecting amplification during or after the PCR reaction.
Convection PCR Apparatus Comprising a Sequence-Specific Detection
Unit
[0486] It is a further object of the invention to provide
"sequence-specific detection" as an additional feature of the
multi-stage thermal convection apparatus embodiments described
herein such as the two-stage apparatus. For some applications, it
will be important to detect the PCR product(s) in a
sequence-specific manner, for instance, in embodiments in which a
user wishes to have accurate identification of target amplicon(s)
and/or elimination of false amplicon(s) during or after a PCR
reaction. The sequence-specific detection feature can be useful for
such needs by providing apparatuses and methods for simultaneous
amplification and sequence-specific detection of the PCR product(s)
during or after the PCR reaction.
[0487] In one embodiment, the multi-stage thermal convection PCR
apparatus is a two-stage apparatus as described herein that
includes at least one operably linked sequence-specific detection
unit (e.g., one, two, three or more of such units). The
sequence-specific detection unit typically comprises one or more
hybridization chips such as DNA chip, for example, one, two, three,
four or more of such hybridization chips. The hybridization chip
typically comprises at least one oligonucleotide probe that is
immobilized on a solid substrate (e.g., less than several hundreds
of such oligonucleotide probes such as one, two, three, four or
more of such oligonucleotide probes). In preferred embodiments, the
hybridization chip comprises two or more oligonucleotide probes
with each probe immobilized at a different location on a suitable
solid substrate. The oligonucleotide probe typically has a nucleic
acid sequence that can specifically hybridize to at least one of
the PCR products. Hence, sequence-specific detection of the
amplified PCR product, including allelic discrimination, is
possible.
[0488] In some embodiments, the hybridization chip may be located
inside of the reaction vessel described above, preferably in
contact with the PCR reaction mixture. In such embodiments, the
hybridization chip may be a separate unit that can be introduced
into the reaction vessel, or it can be a part of the reaction
vessel. The hybridization chip may be located anywhere inside of
the reaction vessel, for instance, the side, bottom or top part of
the reaction vessel. In preferred embodiments, the hybridization
chip is located at the bottom of the inside of the reaction vessel
or at the bottom side of the reaction vessel cap 690, e.g., the
bottom end 696 of the optical port 695 as shown in FIGS. 66A-B and
67.
[0489] In other embodiments, the sequence-specific detection unit
including the hybridization chip may be located outside the
reaction vessel as a separate unit.
[0490] In other embodiments, the multi-stage thermal convection PCR
apparatus is a two-stage apparatus that further includes the
operably linked optical detection unit for detecting hybridization
of the PCR product on the hybridization chip. The optical detection
unit typically detects a fluorescence, absorbance or
chemiluminescence signal from the hybridized PCR product as a
function of position within the hybridization chip. In a particular
embodiment, the optical detection unit has a capability of
capturing an image of the hybridization chip.
[0491] Examples of suitable hybridization chips and/or optical
detection units include, but not limited to those described in the
following references: U.S. Pat. Nos. 5,445,934; 5,545,531;
5,744,305; 5,837,832; 5,861,242; 6,579,680; and 7,879,541; as well
as non-US counterpart applications and patents. See also PCT
Publication Nos. WO 2006/082035; and WO 2012/080339; and Maskos, U.
and Southern, E. M., Nucleic Acids Research, 20(7), pp. 1679-1684
(1992).
[0492] In one embodiment, a detectable probe that can generate an
optical signal as a function of the amount of the hybridized PCR
product is introduced to the sample during or after the PCR
reaction, and the optical signal from the detectable probe is
monitored or detected after hybridization to the hybridization
chip. The detectable probe is typically a detectable label that
generates a fluorescence, absorbance or chemiluminescence signal,
or a detectable DNA binding agent that generates an optical signal
or changes its optical property depending on its binding or
non-binding to, or interaction with the hybridized PCR product.
Useful examples of the detectable probe include, but not limited
to, detectable labels that can be incorporated into the primers or
PCR products, intercalating dyes having a property of binding to
double-stranded DNA, and various oligonucleotide probes having
detectable label(s). Suitable detectable probes and labels have
been described above.
[0493] In a particular embodiment, the structure of the optical
detection unit can be the same as or operably similar to any one of
the structures depicted in FIGS. 59A-B, 60A-B, 61-63, 64A-B, and
67. In another particular embodiment, the detector 650 has an
imaging capability.
[0494] The following examples are given for purposes of
illustration only in order that the present invention may be more
fully understood. These examples are not intended to limit in any
way the scope of the invention unless otherwise specifically
indicated.
EXAMPLES
Materials and Methods
[0495] Three different DNA polymerases purchased from Takara Bio
(Japan), Finnzymes (Finland), and Kapa Biosystems (South Africa)
were used to test PCR amplification performance of various
invention apparatuses. Plasmid DNAs comprising various insert
sequences, human genome DNA, and cDNA were used as template DNAs.
The plasmid DNAs were prepared by cloning insert sequences with
different size into pcDNA3.1 vector. The human genome DNA was
prepared from a human embryonic kidney cell (293, ATCC CRL-1573).
The cDNA was prepared by reverse transcription of mRNA extracts
from HOS or SV-OV-3 cells.
[0496] Composition of the PCR mixture was as follows: a template
DNA with different amount depending on experiments, about 0.4 .mu.M
each of a forward and reverse primer, about 0.2 mM each of dNTPs,
about 0.5 to 1 units of DNA polymerase depending on DNA polymerase
used, about 1.5 mM to 2 mM of MgCl.sub.2 mixed in a total volume of
20 .mu.L using a buffer solution supplied by each manufacturer.
[0497] The reaction vessel was made of polypropylene and had
structural features as depicted in FIG. 40A. The reaction vessel
had a tapered cylindrical shape with its bottom end closed and
comprised a cap that fits with the inner diameter of the top end of
the reaction vessel so as to seal the reaction vessel after
introduction of a PCR mixture. The reaction vessel was (linearly)
tapered from the top to the bottom end so that the upper part had a
larger diameter. The taper angle as defined in FIG. 40A was about
4.degree.. The bottom end of the reaction vessel was made flat in
order to facilitate heat transfer from the receptor hole in the
first heat source. The reaction vessel had a length from the top
end to the bottom end of about 22 mm to about 24 mm, an outer
diameter at the bottom end of about 1.5 mm, an inner diameter at
the bottom end of about 1 mm, and a wall thickness of about 0.25 mm
to about 0.3 mm.
[0498] Volume of the PCR mixture used for each reaction was 20
.mu.L. The PCR mixture with 20 .mu.L volume produced a height of
about 12 to 13 mm inside the reaction vessel.
[0499] All the apparatuses used in the examples below were made
operable with a DC power. A rechargeable Li.sup.+ polymer battery
(12.6 V) or a DC power supply was used to operate the apparatus.
The apparatuses used in the examples had 12 (3.times.4), 24
(4.times.6), or 48 (6.times.8) channels that were arranged in an
array format with multiple rows and columns as exemplified in FIG.
30. The spacing between adjacent channels was made as 9 mm. In the
experiments, the reaction vessel(s) containing the PCR mixture
sample was introduced into the channel(s) after the three heat
sources of the apparatus were heated to desired temperatures. The
PCR mixture sample was removed from the apparatus after a desired
PCR reaction time and analyzed with agarose gel electrophoresis
using ethidium bromide (EtBr) as a fluorescent dye for visualizing
amplified DNA bands.
Example 1. Thermal Convection PCR Using the Apparatus of FIG.
5A
[0500] The apparatus used in this example had the structure shown
in FIG. 5A comprising a channel 70, a first chamber 100, a receptor
hole 73, a through hole 71, a first protrusion 33 of the second
heat source 30, and a first protrusion 23 of the first heat source
20. The length of the first and second heat sources along the
channel axis 80 were about 4 mm and about 9.5 mm, respectively. The
first insulator (or first insulating gap) had a length along the
channel axis 80 near the channel region (i.e., within the
protrusion region) of about 1.5 mm. The length of the first
insulator along the channel axis 80 outside the channel region
(i.e., outside the protrusion region) was about 9.5 mm to about 8
mm depending on position. The first chamber 100 was located on the
lower part of the second heat source 30 and had a cylindrical shape
with a length along the channel axis 80 of about 6.5 mm and a
diameter of about 4 mm. The depth of the receptor hole 73 along the
channel axis 80 was about 2.5 mm for the data presented in this
example although it was varied between from about 1.5 mm to about 3
mm. In this apparatus, the channel 70 was defined by the through
hole 71 in the second heat source 30 and the receptor hole 73 in
the first heat source 20. The channel 70 had a tapered cylinder
shape. Average diameter of the channel was about 2 mm with the
diameter at the bottom end (in the receptor hole) being about 1.5
mm. In this apparatus, all the temperature shaping elements
including the first chamber, the receptor hole, the first
insulator, and the protrusions were disposed symmetrically with
respect to the channel axis.
[0501] As presented below, the apparatus used in this example
having the structure shown in FIG. 5A was found to be efficient
enough to amplify from a 10 ng human genome sample (about 3,000
copies) in about 25 min without the gravity tilting angle. For a 1
ng plasmid sample, PCR amplification resulted in a detectable
amplification in as little as about 6 or 8 min. Hence, this is a
good demonstrating example of a symmetric heating structure that
can provide an efficient PCR amplification without using the
gravity tilting angle. As presented in Example 2, this structure
also works better (i.e., faster and more efficient) when the
gravity tilting angle is introduced. However, a small tilting angle
(about 10.degree. to 20.degree. or smaller) can be sufficient for
most applications.
[0502] 1.1. PCR Amplification from Plasmid Samples
[0503] FIGS. 42A-C show PCR amplification results obtained from a 1
ng plasmid DNA template using the three different DNA polymerases
(from Takara Bio, Finnzymes, and Kapa Biosystems, respectively)
described above. The expected size of the amplicon was 349 bp. The
forward and reverse primers used were 5'-GGGAGACCCAAGCTGGCTAGC-3'
(SEQ ID NO: 1) and 5'-CACAGTCGAGGCTGATCAGCGG-3' (SEQ ID NO: 2),
respectively. In FIGS. 42A-C, the left most lane shows DNA size
marker (2-Log DNA Ladder (0.1-10.0 kb) from New England BioLabs)
and lanes 1 to 4 are results obtained with the thermal convection
PCR apparatus at PCR reaction time of 10, 15, 20, and 25 min,
respectively, as denoted on the bottom of each Figure. The
temperatures of the first and second heat sources of the invention
apparatus were set to 98.degree. C. and 62.degree. C.,
respectively. Depth of the receptor hole along the channel axis was
about 2.5 mm. As shown in FIGS. 42A-C, the thermal convection
apparatus yielded an amplified product at the expected size in very
shorter reaction time. PCR amplification reached a detectable level
at about 10 min and became saturated in about 20 or 25 min. As
manifested, the three DNA polymerases were found to be nearly
equivalent to use with the thermal convection PCR apparatus. A
control experiment was also performed using T1 Biometra
Thermocycler from Biometra for the same PCR mixture containing the
same amount of the plasmid template (data not shown). The control
experiment yielded a product band at the expected size with its
intensity similar to that observed at about 20 or 25 min PCR
reaction time with the invention apparatus; however it took about 3
to 4 times longer time to complete the PCR reaction (about 1 hour
30 min including 5 min pre-heating and 10 min final extension).
[0504] FIG. 43 shows another result of thermal convection PCR
obtained using a plasmid template that can yield a 936 bp amplicon.
Amount of the template plasmid used was 1 ng. The forward and
reverse primers used had the sequences as set forth in SEQ ID NOs:
1 and 2, respectively. The temperatures of the first and second
heat sources were set to 98.degree. C. and 62.degree. C.,
respectively. Depth of the receptor hole along the channel axis was
about 2.5 mm. As shown, even a larger amplicon (about 1 kbp) was
successfully amplified in very short reaction time (about 20 to 25
min), demonstrating a wide dynamic range of the invention
apparatus.
[0505] 1.2. Acceleration of PCR Amplification at Elevated
Denaturation Temperature
[0506] The results shown in FIGS. 44A-D demonstrate acceleration of
the thermal convection PCR at elevated denaturation temperatures.
The template used was a 1 ng plasmid that can yield a 349 bp
amplicon. Except for the denaturation temperature, all other
experimental conditions including the template and primers used
were the same as those used for the experiments presented in FIGS.
42A-C and 43. While the temperature of the second heat source was
set to 62.degree. C., the temperature of the first heat source was
increased from 98.degree. C. (FIG. 44A) to 100.degree. C. (FIG.
44B), 102.degree. C. (FIG. 44C), and 104.degree. C. (FIG. 44D). As
shown, increase of the denaturation temperature (i.e., the
temperature of the first heat source) resulted in acceleration of
PCR amplification. The 349 bp product was barely observable at 10
min reaction time when the denaturation temperature was 98.degree.
C. (FIG. 44A). However, the product band became stronger even at 8
min reaction time when the denaturation temperature was increased
to 100.degree. C. (FIG. 44B). When the denaturation temperature was
further increased to 102.degree. C. (FIG. 44C) and 104.degree. C.
(FIG. 44D), the product band became observable in as short as 6 min
reaction time.
[0507] 1.3. PCR Amplification from Human Genome Sample
[0508] FIGS. 45A-B show two examples of thermal convection PCR for
amplification from a human genome sample. Depth of the receptor
hole along the channel axis was about 2.5 mm. Amount of the human
genome template used for each reaction was 10 ng corresponding to
about 3,000 copies only. FIG. 45A shows results for amplification
of a 479 bp segment of GAPDH gene. The forward and reverse primers
used in this experiment were 5'-GGTGGGCTTGCCCTGTCCAGTTAA-3' (SEQ ID
NO: 3) and 5'-CCTGGTGACCAGGCGCC-3' (SEQ ID NO: 4), respectively. In
this experiment, the temperatures of the first and second heat
sources were set to 98.degree. C. and 62.degree. C., respectively.
FIG. 45B shows results for amplification of a 363 bp segment of
.beta.-globin gene. The forward and reverse primers used in this
experiment were 5'-GCATCAGGAGTGGACAGAT-3' (SEQ ID NO: 5) and
5'-AGGGCAGAGCCATCTATTG-3' (SEQ ID NO: 6), respectively. In this
experiment, the temperatures of the first and second heat sources
were changed to 98.degree. C. and 54.degree. C., respectively, to
match for the lower annealing temperatures of the primers used.
[0509] As shown in FIGS. 45A-B, the thermal convection PCR from
about 3,000 copies of human genome samples yielded amplicons with
correct size in very short reaction time. The PCR amplification was
completed in about 25 or 30 min. These results demonstrate that the
thermal convection PCR is fast and very efficient for amplifying
from low copy number samples.
[0510] 1.4. PCR Amplification from Very Low Copies of Human Genome
Sample
[0511] FIG. 46 shows PCR amplification from very low copy number
samples using the invention apparatus. Template sample used was
human genome DNA extracted from 293 cells. The forward and reverse
primers used in this experiment were
5'-ACAGGAAGTCCCTTGCCATCCTAAAAGC-3' (SEQ ID NO: 7) and
5'-CCAAAAGCCTTCATACATCTCAAGTTGGGGG-3' (SEQ ID NO: 8), respectively.
The temperatures of the first and second heat sources were set to
98.degree. C. and 62.degree. C., respectively. Depth of the
receptor hole along the channel axis was about 2.5 mm. Target
sequence was a 241 bp segment of .beta.-actin. PCR reaction time
was 25 min. As denoted on the bottom of FIG. 46, amount of the
human genome sample used for each reaction was decreased
consecutively, starting from 10 ng (about 3,000 copies) to 1 ng
(about 300 copies), 0.3 ng (about 100 copies), and 0.1 ng (about 30
copies). As manifested, the thermal convection PCR yielded
successful PCR amplification from as little as a 30 copy sample (a
weak band was observed as shown).
[0512] 1.5. Temperature Stability and Power Consumption of the
Invention Apparatus
[0513] Temperature stability and power consumption of the invention
apparatus having the structure shown in FIG. 5A were tested. The
apparatus used in this experiment had 12 channels (3.times.4)
disposed 9 mm apart from each other as shown in FIGS. 30 and 33.
The first and second heat sources were each equipped with a NiCr
heating wire (160a-b) that was disposed in between the channels as
shown in FIG. 33. The apparatus also comprised a fan above the
second heat source to provide cooling to the second heat source
when needed. DC power from a rechargeable Li.sup.+ polymer battery
(12.6 V) was supplied to each heating wire and controlled by PID
(proportional-integral-derivative) control algorism so as to
maintain the temperature of each of the two heat sources at a
pre-set target value.
[0514] FIG. 47 shows temperature variations of the first and second
heat sources when target temperatures were set to 98.degree. C. and
64.degree. C., respectively. The ambient temperature was about
25.degree. C. As shown, the two heat sources reached the target
temperatures within less than about 2 min. During about 40 min time
span after reaching the target temperatures, the temperatures of
the two heat sources were maintained stably and accurately at the
target temperatures. Average of the temperature of each heat source
during the 40 min time span was within about .+-.0.05.degree. C.
with respect to each target temperature. Temperature fluctuations
were also very small, i.e., standard deviation of the temperature
of each heat source was within about .+-.0.06.degree. C.
[0515] FIG. 48 shows power consumption of the invention apparatus
having 12 channels. As shown, the power consumption was high in the
initial time period (i.e., up to about 2 min) in which rapid
heating to the target temperatures took place. After the two heat
sources reached the target temperatures (i.e., after about 2 min),
the power consumption was reduced to lower values. The large
fluctuations observed after about 2 min are result of active
control of the power supply to each heat source. Due to such active
power control, the temperatures of the two heat sources can be
maintained stably and accurately at the target temperatures as
shown in FIG. 47. Average of the power consumption in the
temperature maintaining region (i.e., after about 2 min) was about
4.6 W as denoted in FIG. 48. Therefore, power consumption per each
channel or each reaction was less than about 0.4 W. Since about 25
min to 30 min or less time is sufficient for PCR amplification in
the invention apparatus, energy cost for completion of one PCR
reaction is only about 600 J to 700 J or less as is equivalent to
energy needed to heat up about 2 mL water from room temperature to
about 100.degree. C. one time.
[0516] Invention apparatuses having 24 and 48 channels were also
tested (data not shown). Average power consumption was about 6 to 8
W for the 24 channel apparatus and about 9 to 12 W for the 48
channel apparatus. Hence, power consumption per each PCR reaction
was found to be even less for lager apparatuses, i.e., about 0.3 W
for the 24 channel apparatus and about 0.2 W for the 48 channel
apparatus.
Example 2. Thermal Convection PCR Using the Apparatus of FIG.
11A
[0517] In this example, effect of the gravity tilting angle
.theta..sub.g to the thermal convection PCR was examined. The
apparatus used in this example had the same structure and
dimensions as that used in Example 1 except for incorporation of
the gravity tilting angle .theta..sub.g as defined in FIG. 11A. The
apparatus was equipped with an inclined wedge on the bottom so that
the channel axis was tilted by .theta..sub.g with respect to the
direction of gravity.
[0518] As presented below, introduction of the gravity tilting
angle caused the convective flow faster and thus accelerated the
thermal convection PCR. It was therefore confirmed that a
structural element such as a wedge or leg, or an inclined or tilted
channel that can impose a gravity tilting angle to the apparatus or
the channel is a useful structural element in constructing an
efficient and fast thermal convection PCR apparatus.
[0519] 2.1. PCR Amplification from Plasmid Sample
[0520] FIGS. 49A-E show results of thermal convection PCR as a
function of the gravity tilting angle for amplification from a
plasmid sample. The temperatures of the first and second heat
sources were set to 98.degree. C. and 64.degree. C., respectively.
Depth of the receptor hole along the channel axis was about 2.5 mm.
Amount of the template plasmid used for each reaction was 1 ng. The
primers used had the sequences as set forth in SEQ ID NOs: 1 and 2.
The expected size of the amplicon was 349 bp. FIG. 49A shows
results obtained at zero gravity tilting angle. FIGS. 49B-E show
results obtained at .theta..sub.g equal to 10.degree., 20.degree.,
30.degree., and 45.degree., respectively. At zero gravity tilting
angle (FIG. 49A), the amplified product was barely observable at 15
min reaction time and became strong at 20 min. In contrast, the
amplified product was observable with a significant intensity at 15
min reaction time when the gravity tilting angle of 10.degree. was
introduced (FIG. 49B). Further increase of the product band
intensity at 15 min reaction time and appearance of a weak product
band at a shorter time (i.e., 10 min) were evident as the gravity
tilting angle was increased to 20.degree. (FIG. 49C). Above
20.degree. tilting angle (FIGS. 49D-E), amplification speed was
observed to be similar to that observed at 20.degree. (i.e., only
slightly increased).
[0521] FIGS. 50A-E show another example for amplification of an
about 1 kbp amplicon from a plasmid sample. All the experimental
conditions including the primers used (except for the template
plasmid) are the same as the experiments shown in FIGS. 49A-E. The
expected size of the amplicon was 936 bp. FIG. 50A shows results
obtained at zero gravity tilting angle. FIGS. 50B-E show results
obtained at .theta..sub.g equal to 10.degree., 20.degree.,
30.degree., and 45.degree., respectively. At zero gravity tilting
angle (FIG. 50A), a weak product band was observed at 20 min
reaction time. In contrast, the amplified product was observable at
15 min reaction time when the gravity tilting angle of 10.degree.
was introduced (FIG. 50B). Further increase of the product band
intensity at 15 min reaction time and appearance of a very weak
product band at a shorter time (i.e., 10 min) were observed as the
gravity tilting angle was increased to 20.degree. (FIG. 50C). Above
20.degree. tilting angle (FIGS. 50D-E), only a slight increase of
the amplification speed was observed as compared to the 20.degree.
tilting angle. The effect of the gravity tilting angle observed for
a longer amplicon in this example was found to be similar to the
results obtained for a shorter amplicon shown in FIGS. 49A-E.
[0522] 2.2. PCR Amplification from Various Plasmid Sample
[0523] FIG. 51 shows results of thermal convection PCR
amplification obtained from various plasmid templates with amplicon
size between about 150 bp to about 2 kbp when the gravity tilting
angle of 10.degree. was introduced. The temperatures of the first
and second heat sources were set to 98.degree. C. and 64.degree.
C., respectively. Depth of the receptor hole along the channel axis
was about 2.5 mm. Amount of the template plasmid used for each
reaction was 1 ng. The forward and reverse primers used had the
sequences as set forth in SEQ ID NOs: 1 and 2, respectively. The
expected size of the amplicon was 153 bp for lane 1; 349 bp for
lane 2; 577 bp for lane 3; 709 bp for lane 4; 936 bp for lane 5;
1,584 bp for lane 6; and 1,942 bp for lane 7. PCR reaction time was
25 min for lanes 1-6 and 30 min for lane 7. As shown, nearly
saturated product bands were observed for all amplicons in a short
reaction time. This result demonstrates that thermal convection PCR
is not only fast and efficient, but also has a wide dynamic
range.
[0524] 2.3. PCR Amplification from Human Genome Sample
[0525] FIGS. 52A-E show an example that demonstrates the effect of
the gravity tilting angle for amplification from a human genome
sample. In this experiment, a 10 ng human genome sample (about
3,000 copies) was used as a template DNA. The forward and reverse
primers used in this experiment were
5'-GCTTCTAGGCGGACTATGACTTAGTTGCG-3' (SEQ ID NO: 9) and
5'-CCAAAAGCCTTCATACATCTCAAGTTGGGGG-3' (SEQ ID NO: 8), respectively.
A 521 bp segment of .beta.-actin gene was the target. Other
experimental conditions were the same as those used for the
experiment presented in FIGS. 49A-E and 50A-E above. FIGS. 52A-E
show results obtained when .theta..sub.g was set to 0.degree.,
10.degree., 20.degree., 30.degree., and 45.degree., respectively.
As shown in FIG. 52A, no product band was observed even after 30
min reaction time when no gravity tilting angle was used. In
contrast, the product band was observed in as little as 20 min
reaction time when the gravity tilting angle was introduced (FIGS.
52B-E). Increase of the PCR amplification speed as compared to the
zero tilting angle was observed to be similar for the different
gravity tilting angles examined (i.e., between about 10.degree. to
45.degree.). Only a slight increase of the PCR speed was observed
above 10.degree..
[0526] 2.4. PCR Amplification from Various Target Genes of Human
Genome
[0527] FIGS. 53A-B show further examples of thermal convection PCR
amplification from a human genome sample when the gravity tilting
angle of 10.degree. was introduced. In these examples, a 10 ng
human genome (about 3,000 copies) was used as a template DNA and
primers having relatively low melting temperatures (about
54.degree. C.) as compared to the primers used in other examples
were used. The temperatures of the first and second heat sources
were set to 98.degree. C. and 54.degree. C., respectively. Depth of
the receptor hole along the channel axis was about 2.5 mm. FIG. 53A
shows amplification results for a 200 bp segment of .beta.-globin
gene. The forward and reverse primers used had sequences
5'-CCCATCACTTTGGCAAAGAATTCA-3' (SEQ ID NO: 10) and
5'-GAATCCAGATGCTCAAGGCC-3' (SEQ ID NO: 11), respectively. FIG. 53B
shows amplification results for a 514 bp segment of .beta.-actin
gene. The forward and reverse primers used had sequences
5'-TTCTAGGCGGACTATGACTTAGTTGCG-3' (SEQ ID NO: 12) and
5'-AGCCTTCATACATCTCAAGTTGGGGG-3' (SEQ ID NO: 13), respectively. As
shown in FIGS. 53A-B, the thermal convection PCR yielded very fast
amplification for both genes, delivering significant product band
intensity in as short as 20 min. In the case of the .beta.-actin
sequence, a weak band was observed even at 15 min reaction
time.
[0528] FIG. 54 shows further examples of thermal convection PCR
amplification from 10 ng human genome or cDNA samples when the
gravity tilting angle was 10.degree.. The temperatures of the first
and second heat sources were set to 98.degree. C. and 64.degree.
C., respectively. Depth of the receptor hole along the channel axis
was about 2.5 mm. PCR reaction time was 25 min for lanes 10, 11,
and 13 and 30 min for other lanes. As shown, all fourteen gene
segments with their size ranging from about 100 bp to about 500 bp
were successfully amplified in 25 or 30 min reaction time. Target
genes and corresponding primer sequences are summarized in Table 2
below. Templates used were human genome DNA (10 ng) for lanes 2,
4-7, and 10-14; and cDNA (10 ng) for lanes 1, 3, 8, and 9. The cDNA
samples were prepared by reverse transcription of mRNA extracts
from HOS (lanes 1 and 8) or SK-OV-3 (lanes 3 and 9) cells.
[0529] Table 2. Primer Sequences and Target Genes Used for the
Experiments in FIG. 54
TABLE-US-00002 TABLE 2 Primer Sequences and Target Genes Used for
the Experiments in FIG. 54 Lane Target Amplicon SEQ ID No. Gene
Size NO Primer Sequence 1 p53 123 bp 14
5'-TGCCCAACAACACCAGCTCCTCT-3' 15 5'-CCAAGGCCTCATTCAGCTCTCGGAAC-3' 2
HER2 144 bp 16 5'-CCCCAGCCCTCTGACGTCC-3' 17
5'-TCCGTTTCCTGCAGCAGTCTCCG-3' 3 HER2 192 bp 18
5'-AGCACTGGGGAGTCTTTGTGGATTCTGAG-3' 19
5'-GGGACAGTCTCTGAATGGGTCGCTTTTGT-3' 4 MTHFR 198 bp 20
5'-TGAAGGAGAAGGTGTCTGCGGG-3' 21 5'-AGGACGGTGCGGTGAGAGTG-3' 5 PIGR
216 bp 22 5'-GGGTCCCGCGATGTCAGCCTAG-3' 23
5'-TTCTCCGAGTGGGGAGCCTT-3' 6 .beta.-actin 236 bp 24
5'-ACAGGAAGTCCCTTGCCATCC-3' 13 5'-AGCCTTCATACATCTCAAGTTGGGGG-3' 7
GNB3 268 bp 25 5'-TGACCCACTTGCCACCCGTGC-3' 26
5'-GCAGCAGCCAGGGCTGGC-3' 8 CDK4 284 bp 27
5'-GGTGTTTGAGCATGTAGACCAGGACCTAAGGA-3' 28
5'-GAACTTCGGGAGCTCGGTACCAGAGTG-3' 9 CD24 330 bp 29
5'-TCCAAGCACCCAGCATCCTGCTAG-3' 30
5'-TGGGGAAATTTAGAAGACGTTTCTTGGCCTGA-3' 10 CR2 405 bp 31
5'-GGGAGGTTGGGGTCTTGCCTTTCTG-3' 32 5'-CACCTGTGCTAGACGGTGTTAGCAGC-3'
11 PIGR 433 bp 33 5'-GCCACCTACTACCCAGAGGCATTGTG-3' 34
5'-TGATGGTCACCGTTCTGCCCAGG-3' 12 GAPDH 479 bp 3
5'-GGTGGGCTTGCCCTGTCCAGTTAA-3' 4 5'-CCTGGTGACCAGGCGCC-3' 13
.beta.-globin 500 bp 35 5'-CTAAGCCAGTGCCAGAAGAGCCAAGGAC-3' 36
5'-GCATCAGGAGTGGACAGATCCCCAAAGG-3' 14 .beta.-actin 514 bp 12
5'-TTCTAGGCGGACTATGACTTAGTTGCG-3' 13
5'-AGCCTTCATACATCTCAAGTTGGGGG-3'
[0530] Abbreviations used in Table 2 are as follows. HER2: ERBB2,
v-erb-b2 erythroblastic leukemia viral oncogene homolog 2; MTHFR:
5,10-methylenetetrahydrofolate reductase (NADPH); PIGR: polymeric
immunoglobulin receptor; GNB3: guanine nucleotide binding protein,
beta polypeptide 3; CDK4: cyclin-dependent kinase 4; CR2:
complement receptor 2; GAPDH: glyceraldehydes 3-phosphate
dehydrogenase.
[0531] 2.5. PCR Amplification from Very Low Copies of Human Genome
Sample
[0532] FIG. 55 shows results of thermal convection PCR
amplification from very low copy human genome samples when the
gravity tilting angle was used. The primers used had the sequences
as set forth in SEQ ID NOs: 7 and 8. The amplification target was a
241 bp segment of .beta.-actin gene. The temperatures of the first
and second heat sources were set to 98.degree. C. and 64.degree.
C., respectively. Depth of the receptor hole along the channel axis
was about 2.5 mm. The gravity tilting angle was set to 10.degree.
and the PCR reaction time was set to 25 min. As denoted on the
bottom of FIG. 55, amount of the human genome sample used for each
reaction was decreased consecutively, starting from 10 ng (about
3,000 copies) to 1 ng (about 300 copies), 0.3 ng (about 100
copies), and 0.1 ng (about 30 copies). As manifested, the thermal
convection PCR yielded successful PCR amplification from as little
as a 30 copy sample
[0533] The results presented in this example demonstrate that the
gravity tilting angle is an important structural element that can
be used to increase the speed of the thermal convection PCR.
Moreover, the results suggest that there may be certain limitations
(other than the apparatus itself) in speeding up the thermal
convection PCR. For instance, the speed of the thermal convection
PCR was observed to be about the same when the gravity tilting
angle was larger than about 10.degree. or 20.degree. (e.g., see
FIGS. 49B-E, 50B-E, and 52B-E). These results demonstrate that the
ultimate speed of the thermal convection PCR can be limited by
other factors such as the polymerization speed of the DNA
polymerase and the property of the target template although the
convection speed of the invention apparatus can be increased as
fast as desired.
Example 3. Thermal Convection PCR Using Apparatuses Having
Structural Asymmetry
[0534] Two types of apparatuses were used in this example. The
first apparatus used in this example had the same structure as that
used in Example 1 (i.e., the structure shown in FIG. 5A), but with
slightly different dimensions. The first insulator had a smaller
length along the channel axis 80 near the channel region as
compared to the apparatus used in Example 1. The length along the
channel axis 80 near the channel region (i.e., within the
protrusion region) was about 0.5 mm that was smaller than the about
1.5 mm length of the apparatus used in Example 1. The length of the
first insulator along the channel axis 80 outside the channel
region (i.e., outside the protrusion region) was the same (i.e.,
about 9.5 mm to about 8 mm depending on position). The length of
the first and second heat sources along the channel axis 80 were
about 4 mm and about 11.5 mm, respectively. The first chamber 100
was located on the lower part of the second heat source 30 as shown
in FIG. 5A and had a cylindrical shape with a length along the
channel axis 80 of about 7.5 mm and a diameter of about 4 mm. The
depth of the receptor hole 73 along the channel axis 80 was about
2.5 mm for the data presented in this example although it was
varied between from about 1.5 mm to about 3 mm. The channel 70 had
a tapered cylinder shape with an average diameter of about 2 mm and
the diameter at the bottom end (in the receptor hole) of about 1.5
mm. In this apparatus, all the temperature shaping elements
including the first chamber, the receptor hole, the first
insulator, and the protrusions of the first and second heat sources
were disposed symmetrically with respect to the channel axis.
[0535] The second apparatus used had an asymmetric chamber having a
structure shown in FIG. 20A. The first chamber 100 located on the
lower part of the second heat source was off-centered with respect
to the channel axis by about 0.8 mm as shown in FIG. 20A. Hence,
the first protrusion 33 of the second heat source was also
off-centered with respect to the channel axis by 0.8 mm. Other
structures and dimensions of the second apparatus were identical to
those of the first apparatus described above. In the second
apparatus, the first chamber 100 and the first protrusion 33 of the
second heat source were disposed asymmetrically (i.e.,
off-centered) with respect to the channel axis, while the receptor
hole in the first heat source and the through hole in the second
heat source were disposed symmetrically with respect to the channel
axis.
[0536] As presented below, presence of the structural asymmetry was
found to increase the speed of the thermal convection PCR
substantially. Hence, it is demonstrated that the asymmetric
structural elements such as asymmetric chamber, asymmetric receptor
hole, asymmetric thermal brake, asymmetric insulator, asymmetric
protrusions, etc. are useful structural elements. Such asymmetric
structural elements can be used alone or in combination with other
temperature shaping elements and/or the gravity tilting angle to
modulate (typically to increase) the speed of the thermal
convection PCR as desired.
[0537] 3.1. PCR Amplification from Plasmid Sample
[0538] Template DNA used in this example was a 1 ng plasmid DNA.
Two primers having the sequences as set forth in SEQ ID NOs: 1 and
2 were used. The expected size of the amplicon was 349 bp. The
temperatures of the first and second heat sources were set to
98.degree. C. and 64.degree. C., respectively. No gravity tilting
angle was introduced.
[0539] FIG. 56A shows the results obtained with the first apparatus
having all the temperature shaping elements that are disposed
symmetrically with respect to the channel axis. As shown, a very
weak product band was observed at 15 min reaction time and strong
bands were observed after 20 min.
[0540] FIG. 56B show the results obtained with the second apparatus
that had the asymmetric chamber structure. As described above, the
first chamber was off-centered by about 0.8 mm with respect to the
channel axis. As shown in FIG. 56B, the PCR amplification became
faster and more efficient as compared to the results obtained with
the symmetric apparatus (FIG. 56A). A weak product band was
observed even at 10 min reaction time, demonstrating reduction of
the PCR reaction time by about 5 to 10 min. As manifested, the
small horizontal asymmetry in the first chamber was sufficient to
accelerate the thermal convection PCR dramatically.
[0541] 3.2. PCR Amplification from Human Genome Sample
[0542] FIGS. 57A-B and 58A-B show the results obtained for two
human genome targets, a 241 bp segment of .beta.-actin and a 216 bp
segment of PIGR, respectively. Primers used for the results shown
in FIGS. 57A-B had the sequences as set forth in SEQ ID NOs: 7 and
8. Primers used for the results shown in FIGS. 58A-B had the
sequences as set forth in SEQ ID NOs: 22 and 23. Amount of the
human genome sample used for each reaction was 10 ng corresponding
to about 3,000 copies.
[0543] As shown in FIGS. 57A-B for amplification of the
.beta.-actine sequence, the second apparatus comprising the
asymmetric heating structure (i.e., having the off-centered first
chamber) delivered faster and more efficient PCR amplification
(FIG. 57B) as compared to the first apparatus having the symmetric
heating structure (FIG. 57A). A weak product band was observed at
25 min reaction time when the symmetric heating structure was used
(FIG. 57A). However, when the asymmetric chamber structure was used
(FIG. 57B), the product band became much stronger at the same 25
min reaction time and it became observable at 20 min.
[0544] As shown in FIGS. 58A-B, similar results were obtained when
the target was changed to the PIGR sequence. With the symmetric
heating structure (FIG. 58A), the product was observed as a weak
band at 25 min. However, with the asymmetric chamber structure
(FIG. 58B), the product band became saturated at the same 25 min
reaction time and it became observable as a weak band at 20
min.
[0545] The disclosures of all references mentioned herein
(including all patent and scientific documents) are incorporated
herein by reference. The invention has been described in detail
with reference to particular embodiments thereof. However, it will
be appreciated that those skilled in the art, upon consideration of
this disclosure, may make modifications and improvements within the
spirit and scope of the invention.
Sequence CWU 1
1
36121DNAArtificial Sequenceplasmid forward primer 1gggagaccca
agctggctag c 21222DNAArtificial Sequenceplasmid reverse primer
2cacagtcgag gctgatcagc gg 22324DNAArtificial SequenceGAPDH forward
primer 3ggtgggcttg ccctgtccag ttaa 24417DNAArtificial SequenceGAPDH
reverse primer 4cctggtgacc aggcgcc 17519DNAArtificial
Sequencebeta-globin forward primer 5gcatcaggag tggacagat
19619DNAArtificial Sequencebeta-globin reverse primer 6agggcagagc
catctattg 19728DNAArtificial Sequencebeta-actin forward primer
7acaggaagtc ccttgccatc ctaaaagc 28831DNAArtificial
Sequencebeta-actin reverse primer 8ccaaaagcct tcatacatct caagttgggg
g 31929DNAArtificial Sequencebeta-actin forward primer 9gcttctaggc
ggactatgac ttagttgcg 291024DNAArtificial Sequencebeta-globin
forward primer 10cccatcactt tggcaaagaa ttca 241120DNAArtificial
Sequencebeta-globin reverse primer 11gaatccagat gctcaaggcc
201227DNAArtificial Sequencebeta-actin forward primer 12ttctaggcgg
actatgactt agttgcg 271326DNAArtificial Sequencebeta-actin reverse
primer 13agccttcata catctcaagt tggggg 261423DNAArtificial
Sequencep53 forward primer 14tgcccaacaa caccagctcc tct
231526DNAArtificial Sequencep53 reverse primer 15ccaaggcctc
attcagctct cggaac 261619DNAArtificial SequenceHER2 forward primer
16ccccagccct ctgacgtcc 191723DNAArtificial SequenceHER2 reverse
primer 17tccgtttcct gcagcagtct ccg 231829DNAArtificial SequenceHER2
forward primer 18agcactgggg agtctttgtg gattctgag
291929DNAArtificial SequenceHER2 reverse primer 19gggacagtct
ctgaatgggt cgcttttgt 292022DNAArtificial SequenceMTHFR forward
primer 20tgaaggagaa ggtgtctgcg gg 222120DNAArtificial SequenceMTHFR
reverse primer 21aggacggtgc ggtgagagtg 202222DNAArtificial
SequencePIGR forward primer 22gggtcccgcg atgtcagcct ag
222320DNAArtificial SequencePIGR reverse primer 23ttctccgagt
ggggagcctt 202421DNAArtificial Sequencebeta-actin forward primer
24acaggaagtc ccttgccatc c 212521DNAArtificial SequenceGNB3 forward
primer 25tgacccactt gccacccgtg c 212618DNAArtificial SequenceGNB3
reverse primer 26gcagcagcca gggctggc 182732DNAArtificial
SequenceCDK4 forward primer 27ggtgtttgag catgtagacc aggacctaag ga
322827DNAArtificial SequenceCDK4 reverse primer 28gaacttcggg
agctcggtac cagagtg 272924DNAArtificial SequenceCD24 forward primer
29tccaagcacc cagcatcctg ctag 243032DNAArtificial SequenceCD24
reverse primer 30tggggaaatt tagaagacgt ttcttggcct ga
323125DNAArtificial SequenceCR2 forward primer 31gggaggttgg
ggtcttgcct ttctg 253226DNAArtificial SequenceCR2 reverse primer
32cacctgtgct agacggtgtt agcagc 263326DNAArtificial SequencePIGR
forward primer 33gccacctact acccagaggc attgtg 263423DNAArtificial
SequencePIGR reverse primer 34tgatggtcac cgttctgccc agg
233528DNAArtificial Sequencebeta-globin forward primer 35ctaagccagt
gccagaagag ccaaggac 283628DNAArtificial Sequencebeta-globin reverse
primer 36gcatcaggag tggacagatc cccaaagg 28
* * * * *